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Functions of the Edinger-Westphal Nucleus

Paul D.R.Gamlin

Vision Science Research Center, 626 Worrell Building, University of Alabama

at Birmingham, Birmingham, Alabama 35294, USA

The Edinger-Westphal nucleus (EW), the parasympathetic component of the oculomotor nuclear complex controls several ocular functions by way of its projection to the postganglionic neurons of the ciliary ganglion. These neurons in turn innervate the iris, the ciliary muscle, and the smooth muscle of choroidal blood vessels. It has been shown in both mammals and birds that the EW controls pupilloconstriction and accommodation. It has also been shown in birds, but not so clearly in mammals, that the EW controls choroidal blood flow. Recent studies in primates and pigeons have revealed much about the physiology and anatomy underlying the control of these intraocular functions by the EW. Electrical microstimulation studies of the EW and the intracranial portion of the third nerve in alert primates have confirmed earlier reports that electrical stimulation of the EW elicits pupilloconstriction and ocular accommodation. These studies have also allowed the characteristics and latencies of these two responses to be more precisely described. Single-unit recordings from preganglionic EW neurons related to either pupil diameter or to ocular accommodation have permitted a detailed description of their relationship to these two ocular functions. Further, in birds, electrical microstimulation and lesion studies of the EW have shown that it modulates choroidal blood flow. Anatomical studies in the pigeon have revealed three distinct subdivisions of the EW that control respectively, pupilloconstriction, ocular accommodation, and choroidal blood flow. These subdivisions are selectively innervated by separate afferent pathways related to these three functions. To date, comparable studies have not been conducted in primates. Future studies of the functions of the vertebrate EW can be expected to build upon the electrophysiological approach that has been so successful in primates and the anatomical approach that has been so successful in pigeons.

KEY WORDS: pupillary light reflex; ocular accommodation; choroidal blood flow; ciliary ganglion

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INTRODUCTION

The Edinger-Westphal nucleus (EW) is a distinct nucleus lying immediately dorsal to the somatic subdivisions of the oculomotor complex. It was first described in a developmental study of human neuroanatomical material by Edinger (1885) and, a short time afterward, in a neuropathological study by Westphal (1887). While both authors recognised this

Correspondence: Dr. P.D.R.Gamlin, Vision Science Research Center, 626 Worrell Building, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Tel: +1 205 934-0322; Fax: +1 205 934-5725.

cytoarchitecturally distinct nucleus located dorsal to the oculomotor nucleus, the study by Westphal additionally led to the suggestion that the EW was involved in the innervation of the iris and possibly other intraocular muscles. This suggestion was made because Westphal studied tissue from an individual who, while alive, had been diagnosed with complete external ophthalmoplegia but whose pupils had constricted for near vision and was thus presumed to have an intact pupillomotor nucleus. Westphal found that the neurons of the large-celled somatic oculomotor nucleus had degenerated, and he correlated this loss with the observed external ophthalmoplegia. However, he found sparing of the neurons of the smaller-celled dorsal nucleus (now known as the EdingerWestphal nucleus), and he correlated these spared neurons with the spared pupillomotor function.

Since these pioneering studies, additional studies have clearly shown in many vertebrate classes that the EW is the preganglionic, parasympathetic component of the oculomotor nuclear complex, and is the central source of parasympathetic innervation of the iris, the ciliary body, and certain additional intraocular muscles and tissues. However, in some species, the cells of the cytoarchitecturally-defined EW are not preganglionic neurons but instead have central projections while preganglionic neurons are actually located outside of the EW. For example, cells in the EW of cats are reported to project to the spinal cord and cerebellum (Sugimoto, Itoh and Mizuno, 1978; Loewy and Saper, 1978; Loewy, Saper and Yamodis, 1978; Burde, Parelman and Luskin, 1982; Roste and Dietrichs, 1988), while the ciliary ganglion receives its central input from a collection of preganglionic cells along the midline of the rostral mesencephalon and in the ventral segmental area (Sugimoto, Itoh and Mizuno, 1977; Toyoshima, Kawana and Sakai, 1980; Kuchiiwa, Kuchiiwa and Nakagawa, 1994). This has led to much confusion in the literature, but this confusion does not extend to studies of primates and birds since in both of these groups of animals there is a close correspondence between the EW and the preganglionic, parasympathetic neurons (e.g. Narayanan and Narayanan, 1976; Akert et al., 1980).

In addition, for studies of the physiology of the neurons of the EW, the alert primate has proved most useful for a number of reasons. Alert primates show robust pupillary and accommodative responses (Gamlin et al., 1994; Gamlin, Zhang and Clarke, 1995) and their neural responses are not affected by anaesthesia as was the case for earlier studies in anaesthetised animals (e.g. Sillito and Zbrozyna, 1970a). Also, for anatomical studies of

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the sources of input to the EW, the primate has yielded more reliable results than those obtained in other mammals such as cats and rabbits. This is because the preganglionic neurons in primates are predominantly confined to the cytoarchitecturally-defined EW and thus anterograde studies documenting inputs to the EW can more reliably be considered as demonstrating inputs to the preganglionic neurons. However, despite these advantages of the primate, the pigeon has proved itself by far the most amenable species for detailed anatomical studies of the sources of input to the vertebrate EW. This is for a number of reasons. First, retrograde pathway tracing studies (Cowan and Wenger, 1968; Narayanan and Narayanan, 1976; Lyman and Mugnaini, 1980) have confirmed that, in contrast to most mammals, the avian EW can be clearly delineated cytoarchitecturally, is the sole source of preganglionic afferents to the ciliary ganglion, and does not project to the spinal cord. Instead, in the pigeon a cell group that lies immediately adjacent to but is clearly distinct from the EW projects to the spinal cord (Cabot, Reiner and Bogan, 1982). Second, the avian midbrain and pretectal nuclei have undergone considerable hypertrophy, and many are more sharply defined cytoarchitecturally than in mammals (Ariens-Kappers, Huber and Crosby, 1936; Kuhlenbeck, 1939). Third, the pigeon is a highly visual animal with well-developed neural circuits for the control of the pupil, ocular accommodation, and choroidal blood flow (Gamlin and Reiner, 1991). The above characteristics have resulted in significant advances being made in our understanding of the central visual pathways involved in the parasympathetic control of these ocular functions in this species (Gamlin and Reiner, 1991). Since the peripheral mechanisms regulating these ocular functions can be expected to be evolutionarily conserved and similar in most vertebrates, findings in the pigeon are very relevant to mammals and other vertebrate classes.

For the reasons described in this introduction, this review will concentrate on recent results in pigeons and primates. These results are combined to present as cohesive a view as is currently possible on the physiology and anatomy of the vertebrate EdingerWestphal nucleus. Results from other species will be considered when they contribute to a specific understanding of a particular function of the EW.

CYTOARCHITECTURE AND PROJECTIONS OF THE

EDINGERWESTPHAL NUCLEUS

BIRDS

In birds, the EW is located dorsolateral to the somatic oculomotor nucleus from which it is clearly distinct (Figure 4.1A). The EW, which is also known in birds and reptiles as the accessory oculomotor nucleus, extends caudally for approximately 700 µm from the rostral pole of the oculomotor complex. It lies close to the midline and is bordered on all sides except ventromedially by the central grey. In transverse section, the nucleus appears elliptical, with a maximum diameter of 700 µm, and is surrounded by a neuropil about 50 µm wide. The neurons in this nucleus are of two major classes: neurons with large spherical or elliptical somata, approximately 19–30 µm in mean diameter, and neurons with smaller, fusiform cell bodies, 14–21 µm in mean diameter (Figure 4.1B). There are

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nucleus, pars dorsalis; OMV=Oculomotor nucleus, pars ventralis. Scale bars, A=500 µm; B=100 µm. (Reprinted from Gamlin et al., 1984, with permission of Wiley-Liss, Inc.).

about 500 of the larger cells located mainly in the lateral portion of the EW and between 700 and 1200 of the smaller cells generally located medially (Gamlin et al., 1984; Reiner et al., 1991). Based on these observations and upon studies of afferents, the avian EW has been divided into two cytoarchitecturally distinct subdivisions, a medial (EWm) and a lateral (EWl) (Gamlin, Reiner and Karten, 1982; Reiner et al., 1983, 1991; Gamlin et al., 1984). The majority of the cells in both subdivisions in chicken give rise to parasympathetic preganglionic fibres to the ciliary ganglion and are retrogradely labelled by horseradish peroxidase (HRP) injections of this ganglion (Narayanan and Narayanan, 1976; Lyman and Mugnaini, 1980). The few cells that are unlabelled by these HRP injections may be local interneurons since they are small and have no described extrinsic projections (Lyman and Mugnaini, 1980).

Figure 4.2 Immunohistochemical staining for enkephalin reveals fluorescently-labelled cap-like or calyciform endings (A) and boutonal (B) endings made by preganglionic neurons on postganglionic neurons within the ciliary ganglion. Scale bars=20 µm. (Modified from Reiner et al., 1983)

In the avian ciliary ganglion, there are two classes of neurons, choroidal and ciliary. They are so named because the choroidal neurons innervate the smooth muscle of choroidal blood vessels (Marwitt, Pilar and Weakly, 1971; Pilar and Turtle, 1982), while the ciliary neurons innervate the ciliary body and the iris sphincter muscle, which in birds are composed of striated muscles (Pilar and Turtle, 1982). In addition, there are two classes of terminals in the ciliary ganglion that arise from EW neurons. These terminals contain both acetylcholine and the peptides substance P and enkephalin (Pilar and Turtle, 1982; Erichsen et al., 1982; Reiner et al., 1991). As shown in Figure 4.2, one type of terminal, the so-called cap-like or calyx endings, envelops a portion of the soma of ciliary neurons. The other type of terminal, the so-called boutonal endings, makes multiple, boutonal synaptic endings on choroidal neurons. Lesions of EWm and EWl differentially

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1982; Gamlin et al., 1984; Gamlin and Reiner, 1991). Abbreviations: AP=Area Pretectalis; EWl=EdingerWestphal nucleus, pars lateralis; EWm=EdingerWestphal nucleus, pars medialis; LRF=lateral mesencephalic reticular formation; MRF=medial mesencephalic reticular formation; SCN=suprachiamatic nucleus. (Reprinted from Gamlin and Reiner, 1991, with permission of Wiley-Liss, Inc.).

PRIMATES

The primate EW lies dorsal to the somatic subdivisions of the oculomotor nucleus (Figure 4.4). It is composed of relatively large spherical and ovoid cells (approximately 25–40 µm in diameter) and other spindle-shaped neurons (15 µm–30 µm in diameter) (e.g. Warwick, 1954). The preganglionic neurons within the EW were initially identified by a retrograde degeneration study (Warwick, 1954). Subsequently these neurons have been identified by retrograde neuroanatomical tracer studies following injections into the ciliary ganglion of either HRP (Burde and Loewy, 1980; Clarke, Coimbra and Alessio,

1985b), [125I] wheat germ agglutinin (WGA) (Akert et al., 1980), WGA-HRP (Sun and May, 1993), or fluorescent tracers (Ishikawa, Sekiya and Kondo, 1990). All of these studies reported that labelled, preganglionic neurons are generally the larger, more spherical neurons of the EW and are only slightly smaller than the somatic motoneurons of the oculomotor complex.

Based on the number of retrogradely labelled cells and the estimated number of parasympathetic axons in the oculomotor nerve, Burde and Loewy (1980) suggested in their study that there were 300–400 preganglionic neurons in EW. However, based on the results of Akert et al. (1980), Ishikawa, Sekiya and Kondo (1990), and upon personal observations, the number of preganglionic neurons in the primate EW is more likely to be in the range of 800–1200. This latter estimate is also more consistent with the results reported above for the pigeon.

Throughout much of the length of the oculomotor nucleus, the EW can be seen as the paired medial visceral cell columns of the oculomotor nuclear group. However, anterior to the oculomotor nucleus, this pair of columns merges along the midline to form a contiguous cell group containing cells that are more fusiform than those in the more posterior regions of the EW. As a consequence, this cephalic extension of the EW has often been considered to be separate from the EW and has been termed the anteromedian nucleus by some authors (e.g. Burde and Loewy, 1980; Ishikawa, Sekiya and Kondo, 1990). However, others consider this region a rostral extension of the EW and identify neurons located more ventrally as the anteromedian nucleus (Warwick, 1954; Akert et al., 1980). Based on the retrograde labelling of preganglionic EW neurons, it is clear that the anteriorly located, spindle-shaped, preganglionic neurons that are labelled are anatomically continuous with labelled neurons within the more caudal regions of the EW and should therefore be included within the boundaries of this nucleus, as was originally suggested by Warwick (1954). This arrangement is shown in Figure 4.5 (Akert et al., 1980) and confirmed by personal observation. As shown in this diagram, the EW in

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Figure 4.4 A photomicrograph of a coronal section through the Rhesus oculomotor nuclear complex stained for Nissl substance, showing preganglionic Edinger-Westphal nucleus neurons (arrow) labelled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) as a result of retrograde transneuronal transport following an intravitreal injection of this tracer. Scale bar=250 µm.

rhesus monkeys is approximately 500 µm in dorsoventral extent, extends anterior to the oculomotor nucleus and caudally to within approximately 500 µm of the most posterior region of the oculomotor nucleus (Akert et al., 1980; Personal Observation). A similar rostrocaudal extent for the preganglionic neurons was also reported by Ishikawa, Sekiya and Kondo (1990), but Burde and Loewy (1980) showed a much more restricted distribution with the cells only extending posteriorly approximately halfway through the extent of the oculomotor nucleus.

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Figure 4.5 (A) Three-dimensional reconstruction of the primate oculomotor complex on the basis of serial sections stained for Nissl substance. (B). Location of

retrogradely labelled cells following injection of [125I]- labelled wheat germ agglutinin into the right ciliary ganglion. Abbreviations: AM=anteromedian nucleus; EW=Edinger-Westphal nucleus; NcIII=oculomotor nucleus; SGC=substantia grisea centralis; III=third ventricle. (Reprinted from Akert et al., 1980, with permission of Elsevier Science NL).

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In addition, these latter two groups of investigators reported a few neurons located between the oculomotor nuclei as an additional source of input to the ciliary ganglion and chose to characterize them as a separate nucleus, Perlia’s nucleus (Burde and Loewy, 1980; Ishikawa, Sekiya and Kondo, 1990). From studies in this laboratory and in the marmoset (Clarke, Coimbra and Alessio, 1985b), it is clear however that there are only a very few preganglionic neurons located between the oculomotor nuclei outside of the cytoarchitectural confines of the EW, and it is not clear that this small number of cells constitutes a separate nucleus. Also, in primates there are a few cells located within the boundaries of the Edinger-Westphal nucleus that are not preganglionic neurons and instead project to the cerebellum and spinal cord (Sekiya, Kawamura and Ishikawa, 1984; May, Porter and Gamlin, 1992). Presumably, some of these cells may relay an efference copy signal related to pupilloconstriction or accommodation to the cerebellum that is required by the oculomotor system.

CATS

In cats, a nucleus identified as the EW is located dorsal to the somatic subdivisions of the oculomotor nucleus, but studies indicate that very little of the projection to the ciliary ganglion arises from this nucleus, arising instead from cells in the central grey and ventral tegmental regions (Sugimoto, Itoh and Mizuno, 1977; Toyoshima, Kawana and Sakai, 1980; Kuchiiwa, Kuchiiwa and Nakagawa, 1994). Indeed in the cat, the majority of the neurons in the EW are reported to project not to the ciliary ganglion but to the spinal cord and cerebellum instead (Sugimoto, Itoh and Mizuno, 1978; Loewy, Saper and Yamodis, 1978; Loewy and Saper, 1978; Roste and Dietrichs, 1988).

RABBITS

In rabbits, as in other mammals, the Edinger-Westphal nucleus lies dorsal to the somatic subdivisions of the oculomotor nucleus but, as in cats, it does not contain many preganglionic neurons. Instead, approximately forty preganglionic neurons are reported to lie in the central grey and the tegmental area ventral to the oculomotor nucleus (Johnson and Purves, 1981). As discussed below with respect to birds and primates, this relatively small number of preganglionic neurons is consistent with the observation that in rabbits the EW predominantly subserves the pupillary light reflex (Johnson and Purves, 1983).

OTHER VERTEBRATE CLASSES

Reptiles

The Edinger-Westphal nucleus is well-developed in the monitor lizard (Barbas-Henry and Lohman, 1988). It is an ovoid nucleus lying immediately ventral to the fourth ventricle and dorsal to the somatic oculomotor complex, and it is composed of small to medium-sized bipolar and multipolar neurons. In addition, based on immunohistochemical staining for cholinergic neurons, an EW nucleus has been reported

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in turtles (Powers and Reiner, 1993). Indeed, according to Barbas-Henry and Lohman (1988), there is a well-defined accessory oculomotor nucleus (Edinger-Westphal nucleus) in all reptilian species studied except for snakes where the nucleus is rudimentary or completely absent presumably due to a decrease in the intrinsic eye muscles.

Fish

The Edinger-Westphal nucleus has been reported in both goldfish and kelp bass (Scherer, 1986; Wathey, 1988; Wathey and Wullimann, 1988). In the kelp bass, the EW lies ventral to the fourth ventricle and dorsolateral to the somatic oculomotor complex with its medial border approximately 340 µm from the midline. It has an extent of approximately 250 µm mediolaterally, 150 µm dorsoventrally, and 250 µm rostrocaudally, and consists of between 60 and 100 medium-sized, multipolar neurons with an average soma size of 20 µm (Wathey, 1988). Since this species lacks a pupillary light reflex, these EW neurons are presumably related to accommodation (Wathey, 1988).

DO ALL EW PREGANGLIONIC NEURONS SYNAPSE IN THE CILIARY

GANGLION?

The ciliary ganglion in birds and mammals contains the soma of postganglionic neurons and synaptic contacts from the axons of preganglionic neurons (e.g. Pilar and Turtle, 1982; May and Warren, 1993). Whilst many studies have reported that all preganglionic neurons synapse in this ganglion in mammals (e.g. Ruskell and Griffiths, 1979; Kuchiiwa, Kuchiiwa and Nakagawa, 1994), there has been some debate as to the existence of a synapse in this ganglion for both pupil-related and accommodation-related preganglionic neurons. For example, based on physiological studies (Westheimer and Blair, 1973) and on retrograde anatomical studies (Jaeger and Benevento, 1980; Parelman, Fay and Burde, 1984) it has been reported that EW neurons do not synapse in the ciliary ganglion but instead have a direct projection to the ciliary muscle. Other reports have disagreed with these studies and there is a growing consensus for the existence of a synapse between the preand postganglionic neurons in the primate ciliary ganglion (for a review see Ruskell, 1990). It appears likely that some retrograde tracer experiments appeared to show direct connections between the preganglionic neurons of the Edinger-Westphal nucleus and their eventual peripheral targets because the tracer was taken up by preganglionic fibres en route to the intraocular ganglion cells that are contained within the accessory ciliary ganglia of primates and other mammals. While some of these accessory ganglia are located extraocularly immediately behind the sclera, others are located in the suprachoroid lamina along the intraocular portions of the ciliary nerves from the iris and ciliary body to the scleral canal (Kuchiiwa, Kuchiiwa and Suzuki, 1989; Kuchiiwa, Kuchiiwa and Nakagawa, 1994). Injections in the vicinity of these intraocular cells would thus have involved preganglionic fibres from EW neurons and would have resulted in routine retrograde labelling of these EW neurons.

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THE ROLE OF THE EW IN PUPILLOCONSTRICTION

When the intensity of light falling on the retina increases, many vertebrates display a reflexive constriction of the sphincter pupillae muscles of the iris that results in pupilloconstriction. This pupillary light reflex is rapid and well-developed in both birds and mammals and has been extensively studied in both. The anatomical substrate of this reflex was investigated for many years by anatomists who were aware that a number of disorders of the nervous system, e.g., tertiary syphillis, trauma, tumours, etc., could affect the pupillary response to light (Lowenstein and Loewenfeld, 1969). These investigations can be broadly categorised as clinicopathological studies in humans, and lesion, electrical stimulation, and single-unit recording studies in experimental animals.

PUPILLARY DEFICITS RESULTING FROM DAMAGE TO THE EW OR

EW EFFERENT PROJECTIONS

Earlier this century, based on experimental lesion studies and clinical studies, it was generally accepted that the final efferent link of the pupillary light reflex consisted of a preganglionic projection from the EW in the midbrain to the ciliary ganglion that, in turn, projected to the sphincter pupillae muscle of the iris (for a complete review of the early literature see Loewenfeld, 1993). Specifically, Bernheimer showed that lesions in the region of the EW resulted in a fixed, dilated pupil ipsilateral to the lesion (Bernheimer, 1909). A later study on primates by Pierson and Carpenter (1974) also showed pupillary deficits following discrete lesions in the area of the anterior Edinger-Westphal nucleus. Similarly, lesions of the EW in birds result in dilated pupils that are unreactive to light and an inability to accommodate (Schaeffel et al., 1990). Pupillary immobility is also associated with third nerve palsies or selective damage to the axons of the preganglionic neurons coursing to the ciliary ganglion, and with damage to the postganglionic fibres which results in Adie’s syndrome (Ponsford, Bannister and Paul, 1982; Thompson, 1987).

PUPILLOCONSTRICTION EVOKED BY ELECTRICAL STIMULATION

OF THE EW OR EW EFFERENT PROJECTIONS

Further support for the course of the efferent parasympathetic pupillary pathway and the importance of the EW in pupilloconstriction in primates came from stereotaxic, electrical stimulation studies in the vicinity of EW that elicited pupilloconstriction as well as accommodation (Bender and Weinstein, 1943; Jampel and Mindel, 1967; Westheimer and Blair, 1973). Other studies in the cat (Pitts, 1967; Sillito and Zbrozyna, 1970b), marmoset (Clarke, Coimbra and Alessio, 1985a), and chicken (Troilo and Wallman, 1987) have shown that electrical stimulation of the Edinger-Westphal nucleus evokes pupilloconstriction and accommodation in these species. Electrical stimulation of the ciliary ganglion or nerves in cats has also been shown to elicit pupilloconstriction and accommodation (Olmsted, 1944; Marg, Reeves and Wendt, 1954; Ripps, Breinin and

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Baum, 1961).

Recently, in the alert primate, we have closely examined the pupilloconstriction evoked by electrical microstimulation of the EW or the pupillomotor fibres of the intracranial

Figure 4.6 A figure showing the pupilloconstriction elicited by electrical microstimulation of the intracranial portion of the third nerve for 100 ms. Note that the pupil begins to constrict approximately 100 ms after the onset of stimulation and reaches maximal constriction at approximately 500 ms after the onset of stimulation. Note the subsequent relatively slow redilation. Scale bar=0.5 mm.

portion of the oculomotor nerve (Clarke and Gamlin, 1995). A microelectrode was lowered under physiological guidance either to the EW or the oculomotor nerve and microstimulation was carried out over a wide range of parameters. Pupil diameter was measured using an ISCAN video-based pupillometer. As shown in Figure 4.6, in response to a brief stimulus train, the pupil constricts with a latency of approximately 100 ms, and peak pupilloconstriction occurs approximately 300–500 ms after stimulation. Pupil diameter then returns to baseline with a time constant of approximately 600 ms. These responses were only elicited from the area dorsal to the oculomotor nucleus and from a localised region of the oculomotor nerve. Stimulation in the oculomotor nucleus at more ventral sites elicited eye movements as did stimulation at many sites within the oculomotor nerve.

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RESPONSES OF EW NEURONS DURING THE PUPILLARY LIGHT

REFLEX

In 1970, Sillito and Zbrozyna recorded the activity of preganglionic, pupillomotor neurons in anaesthetised cats. Because of the effects of the chloralose anaesthesia, the pupils were relatively constricted, but still showed a small light reflex. To overcome these effects of anaesthesia, hypothalamic stimulation was used to elicit a “defence reaction” and hence produce pupil dilatation. Using this approach, the authors found that the baseline level of activity of the pupil-related EW neurons was between 6 and 10 spikes/second and was completely inhibited by hypothalamic stimulation. Maximal pupilloconstriction was seen when the pupil-related EW neurons displayed an activity of approximately 8 spikes/ second, but these neurons also displayed transient firing rates with light “on” of up to 28 spikes/second. Light “off” was observed to produce a postexcitatory depression lasting

Figure 4.7 The response of a pupil-related neuron of the EdingerWestphal nucleus during 0.5 Hz sinusoidal modulations in light intensity and the resultant pupillary responses.

The activity of the neuron is modulated sinusoidally and also shows a phase advance with respect to the pupilloconstriction. Note that the animal maintained fixation of the target for the entire period of the trial.

Abbreviations: HL=Horizontal position of the left eye; VL=Vertical position of the left eye. Scale Bar=1 mm.

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as long as 700 ms. In addition to this study, pupil-related parasympathetic activity has been studied postganglionically in the ciliary nerves of rabbits (Nisida and Okada, 1960; Inoue, 1980), and in the ciliary ganglion of cats (Melnitchenko and Skok, 1970) and rabbits (Johnson and Purves, 1983). The reports of these studies are generally consistent with the results of the study by Sillito and Zbrozyna (1970a).

More recently, in our ongoing studies in alert primates, pupil-related EW neurons have been antidromically identified by electrical stimulation of the intracranial portion of the oculomotor nerve. In all cases antidromic activation was confirmed by collision testing (Schlag and Fuller, 1976). An example of an EW pupillomotor neuron is shown in Figure 4.7. In darkness, the firing rate of the neuron was very low. During sinusoidal modulation of light intensity, the activity of the neuron is modulated sinusoidally and varies from approximately 10 spikes/second at a pupillary diameter of approximately 7 mm to 25 spikes/second for near-maximal pupilloconstriction. This neuron also shows a phase advance with respect to pupilloconstriction. This indicates that, during pupillconstriction, these neural signals show a characteristic pre-emphasis. This will result in the transient increase in muscle force that is required to compensate for the sluggish nature of the iris musculature and its associated tissues. Interestingly, the behaviour of this pupil-related EW neuron is very similar to the luminance neurons of the pretectal olivary nucleus that are presumed to provide EW with input related to the pupillary light reflex (Gamlin, Zhang and Clarke, 1995).

PUPIL-RELATED INPUTS TO THE EW

Once the essential role of the EW in the pupillary light reflex had been established by the early part of this century, investigators began to study the sources of inputs to this nucleus that mediated the reflex. It was soon shown that the central afferent limb of the reflex began with retinal ganglion cell fibres and includes the brachium of the superior colliculus (Karplus and Kreidl, 1913). However, the site of termination of these fibres and their subsequent projections remained unclear until the studies of Magoun and colleagues (Ranson and Magoun, 1933; Magoun and Ranson, 1935a, b, Magoun et al., 1936; Hare, Magoun and Ranson, 1935). These experimenters used localised stimulation and lesioning techniques to follow the trajectory of the reflex pathway and showed for the first time that the pretectum is essential for the integrity of the pupillary light reflex. Since these pioneering studies, most reports have implicated the pretectum in providing the EW with the pupil-related input that mediates the pupillary light reflex. But there has been disagreement as to the precise portion of the mammalian pretectum that projected to the EW (Carpenter and Pierson, 1973; Pierson and Carpenter, 1974; Benevento, Rezak and Santos-Anderson, 1977; Steiger and Buttner-Ennever, 1979; Magnuson, Rezak and Benevento, 1980; Young and Lund, 1994), and some studies have even reported that there was no direct projection from any retinorecipient pretectal nucleus to the EW in the cat (Graybiel and Hartweig, 1974; Berman, 1977), tree-shrew (Weber and Harting, 1980), and rat (Nicholson and Severin, 1981).

Because of the distinctiveness of the avian EW, the avian midbrain, and the pretectal nuclei, our studies in the pigeon have been able to resolve some of these issues. To identify the pupil-related inputs to the EW, it was injected with HRP (Gamlin et al.,

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1984). Following these injections, only one retinorecipient region of the pretectum was found to contain HRP-labelled cells. This was a dorsomedially situated region termed the area pretectalis (AP) that receives contralateral retinal input. Approximately 100–250 labelled cells that varied in shape from fusiform (15–20 µm in length) to spherical (12–15 µm in diameter) were observed in the AP contralateral to the injection site. Very few labelled neurons were present ipsilaterally. In order to determine the precise portion of

the EW to which AP projected, this nucleus was injected with [3H]proline/leucine and autoradiographic techniques were used to show that the projection was to the caudolateral pole of the lateral subdivision of the contralateral EW (Figure 4.8). The terminal field was confined to a discrete region of the caudal EW1 approximately 300 µm in rostrocaudal extent and overlying approximately 100 EW neurons. These anatomical results suggest that only a small number of EW cells mediate the pupillary light reflex and are consistent with anatomical studies in the monkey that report that the iris is innervated by only about 3% of ciliary ganglion cells (Warwick, 1954), and with a physiological study in the monkey (Jampel and Mindel, 1967) that reported that few areas in the EW are pupillomotor and that the majority are involved in ocular accommodation.

To establish that this pathway played an essential role in the pupillary light reflex we conducted additional experiments (Gamlin et al., 1984). We showed that unilateral lesions that completely destroyed AP resulted in a fixed, dilated pupil in the eye contralateral to the lesions, while the pupillary light reflex of the eye ipsilateral to the lesioned AP appeared normal. Also, microstimulation of AP was found to elicit a pupillary constriction of the contralateral eye, but no pupillary constriction was evident in the ipsilateral eye. Thus our data for pigeon clearly indicate that a retinorecipient nucleus in the pretectum (area pretectalis) plays a major role in the control of pupilloconstriction. These findings are summarised in Figure 4.9.

To directly relate the findings in the pigeon to those in mammals, the mammalian pretectal nucleus that is comparable to AP had to be identified. Studies in the rat, mouse, rabbit, tree-shrew (Scalia, 1972), cat (Berman, 1977), and monkey (Benevento, Rezak and Santos-Anderson, 1977) show that the pretectal olivary nucleus is retinorecipient and occupies a region in the pretectum that is topographically comparable to that occupied by AP in the pigeon. Fibres within AP stain for substance P, catecholamines and enkephalin (Gamlin et al., 1984). The pretectal olivary nucleus (PON) in the rat also contains substance P (Ljungdahl, Hökfelt and Nilsson, 1978), catecholamines (Lindvall et al., 1974), and enkephalin (unpublished observations) and is thus histochemically similar to AP in the pigeon. Thus, based on topographic and histochemical grounds, the PON of mammals appears to be similar to the AP of birds.

In light of these results in the pigeon and the conflicting results regarding the source of pretectal input to the EW in mammals, we have recently investigated the source of the pretectal input to the EW in the rhesus monkey (Gamlin and Clarke, 1995). To identify the afferent pretectal regions, WGA-HRP was injected into the EW under physiological

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pretectalis. Dorsal is to the top and medial is to the right in each photograph. Abbreviations: CG=central grey; EWl=Edinger-Westphal nucleus, pars lateralis; EWm=Edinger-Westphal nucleus, pars medialis; FRM =Medial mesencephalic reticular formation; OMd=Oculomotor nucleus, pars dorsalis. Scale bar=50 µm. (Reprinted from Gamlin et al., 1984, with permission of Wiley-Liss, Inc.).

Figure 4.9 Schematic illustration of the central course of the pupillary light reflex in the pigeon. Abbreviations: AP=area pretectalis; EW=Edinger-Westphal nucleus; l=pars lateralis of EW; m=pars medialis of EW; TeO=Optic tectum; V=Ventricle. (Reprinted from Gamlin et al., 1984, with permission of Wiley-Liss, Inc.).

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Figure 4.10 Schematic illustration of the subcortical connections mediating the pupillary light reflex in the primate. Abbreviations: AQ=aqueduct; EW=Edinger-Westphal nucleus; OC=optic chiasm; PC=posterior commissure; PON=pretectal olivary nucleus. (Redrawn from Gamlin and Clarke, 1995).

guidance. Intravitreal injection of the same tracer were also made in other animals to define the retinal terminal fields within the pretectum. Following injection of WGA-HRP in the Edinger-Westphal nucleus and appropriate processing, retrogradely labelled cells were found in only one retinorecipient pretectal nucleus, the pretectal olivary nucleus. Almost all labelled cells were located contralateral to the injection site. Intravitreal injection of tracer resulted in anterograde labelling of all the retinorecipient pretectal nuclei including the pretectal olivary nucleus. The retinal terminal field in the pretectal olivary nucleus coincided with the location of the cells that were retrogradely labelled by the injection of tracer into the Edinger-Westphal nucleus and its vicinity. As summarised in Figure 4.10, these results are very similar to those obtained by us in the pigeon and demonstrate that there is a direct projection from the pretectum to the Edinger-Westphal nucleus, that it arises from only one retinorecipient pretectal nucleus, the pretectal olivary nucleus, and that the pretectal olivary nucleus projects predominantly contralaterally to the EW by way of the posterior commissure.

Figure 4.10 indicates that in primates the pretectal projection to EW is predominantly contralateral and not bilateral, and this contrasts with the generally held textbook view

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(e.g. Thompson, 1987). However, the results of Pasik, Pasik and Bender (1969) are consistent with this proposal. These data show that lesions of the posterior commissure result in bilaterally dilated pupils, which fail to constrict in response to increased illumination, but show a dark reflex. Some clinical evidence in humans (Collier, 1927; Keane and Davis, 1976) also indicates that lesions of the posterior commissure bilaterally abolish the pupillary light reflex, thus supporting the idea that the pretectal projection to the EW is predominantly contralateral in both humans and monkeys. The proposal that the projection from the pretectum to EW is entirely contralateral might also seem incompatible with the consensual pupillary light reflex seen both in normal monkeys and in monkeys with optic tract lesions (Lowenstein, 1954). However, the consensual reflex could be mediated indirectly by way of a projection from the PON to the contralateral PON. Alternatively, since each retina projects bilaterally to the pretectum, the consensual pupillary light reflex could be mediated by this route.

Support for this viewpoint comes from other studies in the monkey that have generally yielded comparable results demonstrating that retrogradely labelled cells in the pretectum are predominantly confined to the contralateral pretectal olivary nucleus after injections of HRP or WGA-HRP into the EW (Steiger and Buttner-Ennever, 1979; Magnuson, Rezak and Benevento, 1980; Buttner-Ennever et al., 1996). However, the results of two recent anterograde studies have raised some questions regarding the details of this proposed pathway. One of these studies investigated the pretectal projection to the EW and suggested that the PON projects not to the EW proper, but immediately lateral to it (Buttner-Ennever et al., 1996). Following intraocular injections of tritiated amino acids, the other study reported transneuronal anterograde labelling over a similar region lateral to the EW proper (Kourouyan and Horton, 1997). Specifically, in both studies, the projection was reported to be to the so-called lateral visceral cell column (Carpenter and Peter, 1970) where, except for a report by Burde and Williams (1989), preganglionic neurons have not been reported. While it is hard to explain these results, it is possible that if the pretectal projection to the EW in primates is as localised as we observed in the pigeon, then the specific region of EW that receives direct pretectal input could have been overlooked in these studies. This would have been particularly likely if the anterograde label in the EW proper was very weak due to the insensitivity of the autoradiographic technique. Alternatively, neurons in the lateral visceral cell column could be interneurons and project to the preganglionic, pupillomotor neurons of the EW. Further study will be required to resolve this issue.

In addition to this pretectal, pupil-related input to the EW, the cerebellum has also been reported to project to the EW (e.g. Thomas et al., 1956; May, Porter and Gamlin, 1992). Electrophysiological and lesion studies in cats (Tsukahara, Kiyohara and Ijichi, 1973; Hultborn, Mori and Tsukahara, 1973, 1978; Ijichi et al., 1977) have reported that this projection may modulate pupillary function. However, as emphasised by Hultborn, Mori and Tsukahara (1978) and as described in the next section, while these papers provide some evidence that the cerebellum modulates the pupillary light reflex, there is even stronger evidence that the cerebellar projection to the EW modulates accommodation.

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THE ROLE OF THE EW IN OCULAR ACCOMMODATION

The basic mechanism of accommodation was first described by Helmholtz (1867). Briefly, changes in focus of the lens of the eye are brought about by changes in force from the ciliary muscle which acts in an antagonistic fashion with the fibres of the zonule. These zonular fibres support the lens capsule and act to maintain the lens in a relatively “flattened” state at rest. Ocular accommodation occurs when ciliary muscle contraction results in a reduction in tension in the zonular fibres which produces a “bulging” (increase in convexity) of the lens and a concomitant increase in its refractive power. Accommodative ranges of up to 20 dioptres, i.e. the ability to focus as close as 5 cm, are seen in primates (e.g. Chin et al., 1968; Crawford, Terasawa and Kaufman, 1989) and birds (e.g. Troilo and Wallman, 1987). The parasympathetic innervation of the ciliary muscle dominates the dynamics of these accommodative responses. Increases in parasympathetic innervation act rapidly (within 1 second) to produce substantial positive accommodation of up to 20 dioptres (e.g. Chin et al., 1968; Tornqvist, 1967; Gamlin et al., 1994), while sympathetic innervation acts with the much slower time course of 10–40 s to produce hyperopia (accommodation for far) of at most 1.5 dioptres (e.g. Tornqvist, 1966, 1967; Gilmartin, 1986).

The parasympathetic innervation for the ciliary muscle arises from postganglionic neurons of the ciliary ganglion and from neurons of the accessory ciliary ganglia (e.g. Kuchiiwa, Kuchiiwa and Suzuki, 1989). In turn, these postganglionic neurons are innervated by accommodation-related neurons of the Edinger-Westphal nucleus. Electrical stimulation and single-unit recording in the EW and its efferent pathways in primates and birds have revealed much about the physiology of these connections. Also, anatomical investigations in birds and primates have revealed much about the sources of afferents to the EW that mediate accommodation.

ACCOMMODATION EVOKED BY ELECTRICAL STIMULATION OF THE

EW OR EW EFFERENT PROJECTIONS

A number of studies have shown that electrical microstimulation in or immediately adjacent to the EW evokes ocular accommodation in primates (Bender and Weinstein, 1943; Jampel and Mindel, 1967; Westheimer and Blair, 1973; Clarke, Coimbra and Alessio, 1985a; Judge and Cumming, 1986; Crawford, Terasawa and Kaufman, 1989; Gamlin et al., 1994), in cats (Pitts, 1967), and in birds (Troilo and Wallman, 1987). These results of EW stimulation are also consistent with studies in which electrical stimulation of the ciliary ganglion or nerves produced similar increases in accommodation in cats (Olmsted, 1944; Marg, Reeves and Wendt, 1954; Ripps, Breinin and Baum, 1961).

More recently we have examined the effects of electrical microstimulation in the EW of the alert rhesus monkey (Figure 4.11; Gamlin et al., 1994). Accommodation was measured by a continuous recording optometer based on a design of Kruger (1979). To facilitate measurement of the latency of the response, both accommodation (ACC) and accommodative velocity (ACCV) are shown in Figure 4.11A. As can be seen in this figure, the ACCV trace crosses the zero velocity line approximately 75 ms after the

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beginning of the stimulus train. In Figure 4.11A, some convergence and adduction of the right eye is visible. However, Figure 4.11B shows that, with shorter stimulation times, specific changes in accommodation could be elicited that were not associated with significant changes in eye position. Repeated measures of the latency of the stimulationinduced responses were made at a number of sites within the EW, and the results from two animals were essentially identical, with accommodative responses being evoked with latencies of 75 ms.

Figure 4.11 Effect of microstimulation of the Edinger-Westphal nucleus on ocular accommodation. (A) shows stimulation (80 ms; 500 Hz; 40 µA) producing an accommodative response with a latency of 75 ms. Accommodative velocity (ACCV) is also shown to facilitate estimation of the latency of the accommodative response. Note that, in addition to accommodation, there is an adduction of the right eye which presumably results from current spread to the nearby medial rectus motoneurons of the “C” subgroup of the right oculomotor nucleus. The stimulation also produces a small amount of convergence that is probably the result of the activation of the axon collaterals of near-response neurons that project to medial rectus motoneurons and presumably also to the Edinger-Westphal nucleus. (B) confirms the specificity of the microstimulation effect by showing that only accommodation is elicited when a stimulation train of shorter duration (10 ms; 500 Hz; 40 µA) is used. Abbreviations: ACC=accommodation; HL=horizontal

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position of the left eye; HR=horizontal position of the right eye; VA=vergence angle. Scale bar=1 meter angle and 1 dioptre.

(Reprinted from Gamlin et al., 1994, with permission of the American Physiological Society).

RESPONSES OF EW NEURONS DURING OCULAR ACCOMMODATION

To examine the single-unit responses of EW neurons during accommodation, preganglionic neurons were identified by antidromic activation from a stimulating electrode placed in the intracranial portion of the ipsilateral oculomotor nerve just as was done to identify pupil-related preganglionic EW neurons. Also, as was the case for pupilrelated preganglionic neurons, antidromic activation was confirmed by collision testing (Schlag and Fuller, 1976). An example of the behaviour of an accommodation-related EW neuron is shown in Figure 4.12 during sinusoidal tracking of a target moving in depth at 0.5 Hz. We found that the behaviour of these neurons during normal binocular viewing was qualitatively the same for all cells. In all cases, their firing rates in darkness

Figure 4.12 Behaviour of a preganglionic EW neuron during sine wave tracking of a target moving in depth. The firing rate modulates between approximately 15 spikes/second and 25 spikes/second for the change in accommodation of 5 dioptres. Note that there is a significant phase lead in the firing rate of this cell with respect to accommodation that cannot be accounted for solely by the latency between the activity of the cell and accommodation. This phase lead results from a substantial component of the firing rate being related to the dynamics of the movement. Abbreviations: ACC=accommodation; HL=horizontal position of the

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left eye; HR=horizontal position of the right eye; VA=Vergence angle. Scale bar=2 metre angles and 2 dioptres. (Reprinted from Gamlin et al., 1994, with permission of the American Physiological Society).

and at optical infinity were low, but all were active (average activity=11.6 spikes/second), and all increased their firing rates with increases in accommodation. On average these neurons showed a sensitivity to accommodation of 3.3 (spikes/second)/dioptre. Also as expected, we found that the firing rate of these EW neurons was unaffected by horizontal conjugate eye movements.

In addition, as shown in this figure, the neuron shows a phase lead that cannot be accounted for solely based on the 75 ms delay between neural activity and ocular accommodation that would have been expected based on the results from electrical microstimulation. Instead, this phase lead results from a significant sensitivity to the speed of accommodation that was seen on this cell and on all the accommodation-related EW neurons that were characterised with sine-wave tracking in depth. Overall, accommodation-related preganglionic neurons were related to the rate of change of accommodation with a sensitivity of 1.2 (spikes/second)/(dioptre/second) at 0.5 Hz. This neural activity is presumably required to provide the additional innervation of the ciliary muscle that is needed to generate sufficient transient force to compensate for the characteristics of this muscle and the peripheral accommodative apparatus, which are presumed to be very sluggish (Gamlin et al., 1994).

Interestingly, the firing rates of EW neurons is extremely low when compared with the firing rates of the neurons related to accommodation and convergence that are encountered around the oculomotor nucleus and that are presumed to provide accommodation-related input to the EW. For example, the highest gain for an identified EW neuron was 6.4 (spikes/second)/dioptre while the lowest gain for a near-response cell identified as projecting to the medial rectus subdivision of the oculomotor nucleus was 12.0 (spikes/ second)/dioptre (Zhang, Mays and Gamlin, 1992). Overall, the gain of identified accommodation-related EW neurons is more than six times lower than that of previously reported midbrain near-response neurons (Mays, 1984; Judge and Cumming, 1986; Zhang, Mays and Gamlin, 1992). The functional significance of this is currently unclear, but may be related to the fact that midbrain near-response neurons innervate medial rectus motoneurons with much higher firing rates (Gamlin and Mays, 1992).

ACCOMMODATION-RELATED INPUTS TO THE EW

There have been few anatomical studies of the inputs to the primate EW that might control accommodation. However, as shown in Figure 4.13, we have used anatomical techniques in the rhesus monkey to show that the fastigial nucleus of the cerebellum projects to the EW (May, Porter and Gamlin, 1992). Also, our electrophysiological studies in the alert rhesus monkey have shown that some neurons in this nucleus are related to accommodation and vergence and presumably modulate the activity of EW neurons by way of this projection (Zhang and Gamlin, 1996). In cats, neurons related to accommodation have been reported in both the fastigial and interpositus nucleus

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In addition to these near-response cells located immediately adjacent to the oculomotor nucleus, other cells that modulate their activity during the near response have been reported approximately 4–6 mm more dorsal in an ill-defined region that includes, or is close to, the nucleus of the posterior commissure (Judge and Cumming, 1986; Mays et al., 1986). On the basis of cytoarchitecture and embryological considerations, the nucleus of the posterior commissure in primates may be considered comparable to the region of the avian mesencephalon which has been termed the lateral mesencephalic reticular formation (LRF) (Kuhlenbeck, 1937; Kuhlenbeck, 1939).

In the rhesus monkey, it is possible that these near-response cells in both the midbrain and pretectal region project monosynaptically to the EW. Although this has not been specifically investigated either physiologically or anatomically in this species, there is support for connections of this nature from some anatomical studies in the pigeon (Gamlin and Reiner, 1991). As described below, in this species it has been shown that there are cells in the medial mesencephalic reticular formation (MRF) that are located in a cytoarchitecturally and topographically similar location to cells immediately adjacent to the oculomotor nucleus in primates. These cells project to the entire EW including its accommodative subdivision, the rostromedial EWl. Also, cells in the LRF that are located in a cytoarchitecturally and topographically similar location as cells in the nucleus of the posterior commissure of primates project specifically to the accommodative subdivision of the EW

To investigate accommodation-related and other inputs to the EW of the pigeon, injections of HRP were placed into it. These injections retrogradely labelled cells in a number of regions including the MRF and LRF. To investigate these apparent sources of input to EW further, a series of anterograde studies were conducted in which animals

received 3H proline/leucine injections in and around the MRF. One of these injections, which is shown in Figure 4.14A, resulted in clear anterograde labelling of the entire contralateral EW. Weaker, but nevertheless clear, anterograde labelling of the entire ipsilateral EW was also present. Both contralaterally and ipsilaterally this anterograde label formed a dust-like pattern over the entire nucleus (Figure 4.15). This terminal labelling may reflect a single afferent input related to accommodation, pupil, and choroidal blood flow. Alternatively, it is possible that this pattern of terminal label in the EW results from the involvement of two afferent systems. It appears as though a rostrolateral region of the MRF projects to the EWl and that this input is related to accommodation and to pupilloconstriction. In addition, a separate and independent pathway, which appears to arise from more medial reticular regions, projects to all EW subdivisions and possibly modulates the overall transmission through this nucleus. As discussed in a subsequent section, this would be consistent with evidence in mammals for a modulatory influence of the reticular formation on transmission through the EW.

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in the Edinger-Westphal nucleus (EW) following the injection of tritiated amino acids into the medial mesencephalic reticular formation that is shown in Figure 4.14A. Dotted lines indicate the boundaries of the EW ipsilateral to the injection site. OMd=Oculomotor nucleus, pars dorsalis. Scale bar=100 µm. (Reprinted from Gamlin and Reiner, 1991, with permission of Wiley-Liss, Inc.).

The possibility that the MRF input to the EW plays both a direct and a modulatory role of the near response is also supported by immunohistochemical studies that have revealed that some MRF cells projecting to the EW stain for the presence of the neuropeptide enkephalin, while others do not (Gamlin and Reiner, 1987; Reiner, unpublished observations). Thus there appear to be two functionally distinct subpopulations of cells in the MRF projecting to EW. One population, immediately adjacent to the oculomotor nucleus controls accommodation and pupilloconstriction, the other, more caudally located in the raphe, projects to all the EW subdivisions and modulates transmission through the EW.

To investigate the apparent projection of the LRF to EW, we carried out a series of

anterograde studies in which animals received [3H]proline/leucine injections in and around this nucleus. One of these injections, which is shown in Figure 4.14B, resulted in clear anterograde labelling of the contralateral EWl (Figure 4.16). Weaker, but nevertheless clear, anterograde labelling of the ipsilateral EWl was also present (Figure 4.16). The pattern of anterograde label was very different from that seen following MRF injections. Instead of being dust-like, the anterograde label was present over large-calibre fibres that terminated specifically in the lateral region of the EW (Figure 4.16). The terminal field overlay the accommodative subdivision of EWl; it was also closely associated with the smaller, pupilloconstrictor subdivision of the EWl, appearing to overlie some cells in this caudolateral subdivision.

Our anterograde studies showed that cells in the LRF project to the accommodative subdivision of EWl. They also showed a projection field closely associated with the localised region of EWl that we have identified as the pupilloconstrictor subdivision. It therefore seems likely that the input to the EWl from the LRF is to both the accommodative and pupillomotor subdivisions and that this pathway may mediate or modulate both accommodation and pupilloconstriction. The results of the above studies are summarised in Figure 4.3.

These results in pigeons suggest that, if the projections in primates are comparable, there are monosynaptic projection from the near response cells in both the supraoculomotor area and the nucleus of the posterior commissure onto the EW. Projections of this nature would presumably mediate or modulate accommodation and possibly the pupillary near response. In mammals, authors have interpreted the projection from the nucleus of the posterior commissure to much of the EW as being involved in the pupillary light reflex (e.g. Benevento, Rezak and Santos-Anderson, 1977). However, since only a small percentage of the EW cells are involved in the pupillary light reflex in both mammals and birds (Warwick, 1954; Gamlin et al., 1984), it is unlikely that this

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THE ROLE OF THE EW IN REGULATING CHOROIDAL BLOOD FLOW

Increases in illumination of the retina places both metabolic and thermoregulatory demands on this structure (Parver et al., 1982; Parver, Auker and Carpenter, 1983). These demands are particularly pronounced for the outer layers of the retina including the photoreceptor layer which are supplied with blood by the choroid. Thus, the choroid is essential to meeting the metabolic and thermoregulatory demands of the outer layers of the retina. Changes in retinal illumination alter these demands and presumably require a compensatory alteration on the part of choroidal blood flow. Indeed, as described below, a centrally-mediated reflex controlling choroidal blood flow appears to play a role in preventing the deleterious effects of light on photoreceptors under normal ambient light levels. Parver and colleagues (1982, 1983) have shown in monkeys and humans that increases in illumination of a given eye result in increased blood flow within both that eye and the contralateral eye. Dysfunctions of this reflex may play a role in at least some classes of degenerative diseases of the retina. In support of this, retinal photoreceptors have been shown to degenerate when the choroidal blood supply is occluded (Goldor and Gay, 1967), or the innervation to the choroid is compromised (Shin, Fitzgerald and Reiner, 1993).

In both birds and mammals, sympathetic innervation of the choroid arises from the superior cervical ganglion and serves to decrease choroidal blood flow (e.g. Bill and Nilsson, 1985; Gherezghiher, Hey and Koss, 1989). In contrast, the parasympathetic input has a vasodilatory action and serves to increase choroidal blood flow in both mammals (Bill and Sperber, 1990) and birds (Fitzgerald, Vana and Reiner, 1990a). In birds, this parasympathetic innervation arises predominantly from the choroidal neurons of the ciliary ganglion (Pilar and Turtle, 1982; Meriney and Pilar, 1987; Cuthbertson et al., 1996), with a lesser contribution from the facial nerve by way of the sphenopalatine ganglion (Cuthbertson et al., 1997). In contrast, based on anatomical studies in mammals the parasympathetic innervation of the choroid is reported to arise predominantly from the sphenopalatine ganglion (Ruskell, 1971), but physiological studies in mammals also suggest a contribution from the oculomotor nerve by way of the ciliary ganglion (Gherezghiser, Hey and Koss, 1989; Nakanome et al., 1995).

DEFICITS IN THE CONTROL OF CHOROIDAL BLOOD FLOW THAT RESULT FROM LESIONS OF THE EW OR EW EFFERENT PROJECTIONS

Under normal conditions pigeons display a stable baseline choroidal blood flow that increases significantly with increases in retinal illumination (Fitzgerald et al., 1996). However, following permanent lesions of the EW, baseline choroidal blood flow is decreased by approximately half and does not increase with increases in retinal illumination (Fitzgerald et al., 1996). Importantly, these permanent lesions of EW in the pigeon result in clear retinal pathology as evidenced by Müller cells expressing increased levels of glial fribrillary acidic protein (Fitzgerald, Vana and Reiner, 1990b). In chicks, when the choroidal nerves are sectioned, the decrease in choroidal blood flow is even

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more significant, decreasing to approximately one fifth of normal, and this results in a histologically verified loss of the cells of the outer retina (Shin, Fitzgerald and Reiner, 1993). To my knowledge, to date, similar experiments have not been conducted in mammals.

CHANGES IN CHOROIDAL BLOOD FLOW EVOKED BY ELECTRICAL STIMULATION OF THE EW OR EW EFFERENT PROJECTIONS

Electrical microstimulation of the Edinger-Westphal nucleus or the ciliary nerves has been shown to evoke increases in choroidal blood flow in the cat (e.g. Gherezghiser, Hey and Koss, 1989; Nakanome et al., 1995) and the rabbit (e.g. Stjernschantz, Alm and Bill,

Figure 4.17 Effects on choroidal blood flow of electrical microstimulation of the Edinger-Westphal nucleus (EW) and suprachiasmatic nucleus (SCN). Horizontal solid bars indicate the timing and duration of electrical stimulation. Choroidal blood flow was measured transsclerally at a site below the superior rectus muscle in an animal deeply anaesthetised with ketamine/xylazine. Scale Bar=50 Blood Flow Units. (Redrawn from Fitzgerald et al., 1996).

1976). It has also been reported that, as a possible consequence of this increase in intraocular blood flow, stimulation of the Edinger-Westphal elicits increases in intraocular pressure in the cat (e.g. Gherezghiser, Hey and Koss, 1990). However, the mechanism underlying this increase in intraocular pressure has not been investigated further.

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injection of tritiated amino acids into the contralateral suprachiasmatic nucleus. Dorsal is to the top and medial is to the right in each photograph. CG=central grey; EWl=Edinger-Westphal nucleus, pars lateralis; FRM=medial reticular formation; OMd=oculomotor nucleus, pars dorsalis. Scale bar=50 µm. (Reprinted from Gamlin et al., 1984, with permission of WileyLiss, Inc.).

Studies of the influence of electrical stimulation of the EW on choroidal blood flow have also been conducted in the pigeon (Fitzgerald, Vana and Reiner, 1990a; Fitzgerald et al., 1996). In these studies, a TSI Laserflo blood perfusion monitor using laser doppler flowmetry was used to measure choroidal blood flow. Measurements were made transsclerally from the vascular beds beneath the superior rectus or superior oblique muscle. Since, the blood flow in these particular vascular beds has not been calibrated with an independent method such as microspheres (Lindsberg et al., 1989), it is presented in arbitrary Blood Flow Units, although these can be expected to correspond quite closely to the units of flow of ml/min/100g tissue. Figure 4.17 shows an example of the clear increase in choroidal blood flow that was evoked by electrical microstimulation (100 Hz, 400 µA) of the EW of the pigeon.

BLOOD FLOW-RELATED INPUTS TO EW

To identify the source of the input to EW that was related to the control of choroidal blood flow, we injected HRP into this nucleus. Retrogradely-labelled cells were found in a small retinorecipient, hypothalamic nucleus in the rostral diencephalon termed the suprachiasmatic nucleus (SCN) (Gamlin, Reiner and Karten, 1982). Using autoradiographic techniques, we confirmed that the SCN projected to the choroidal subdivision of the EW, the EWm (Figure 4.18). In additional studies, we found that the terminals and fibres of this projection contain substance P (Gamlin, Reiner and Karten, 1982). Also, as shown in Figure 4.17, we have found that electrical microstimulation of the SCN elicits increases in choroidal blood flow (Fitzgerald et al., 1996). In addition, lignocaine blockade of the EW can prevent these increases in blood flow that are observed with electrical stimulation of the SCN (Fitzgerald et al., 1996). Thus, on the basis of anatomical, electrical stimulation, and lesion studies this retino-SCN-EWm pathway has been shown to be a significant component of the parasympathetic control of choroidal blood flow in pigeons and it is summarised in Figure 4.19.

As described above, physiological studies suggest that the mammalian EW may be involved with the control of choroidal blood flow as well as with the control of accommodation and pupilloconstriction. However, to date, no study has described a separate choroidal subdivision of the mammalian EW that is equivalent to the avian EWm. This may well result from the fact that all the cells in the mammalian ciliary ganglion innervate smooth muscle and thus the preganglionic neurons providing their input can be expected to appear similar. Separate subdivisions of the EW containing morphologically different preganglionic neurons may be present in birds because cells in

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these two subdivisions innervate cells in the ciliary ganglion that project to different muscle types as peripheral targets. Ciliary neurons innervate striated muscle while choroidal neurons innervate smooth muscle (Pilar and Tuttle, 1982). Thus it is possible that cells in a cytoarchitecturally indistinct region of the mammalian EW are involved with the control of choroidal blood flow. However, to my knowledge, this possibility has not been systematically examined anatomically or physiologically in primates.

Figure 4.19 Schematic illustration of the projection from the suprachiasmatic nucleus (SCN) to the EdingerWestphal nucleus (EW) in the pigeon. Abbreviations: TeO=Optic tectum; V=Ventricle.

MODULATION OF INFORMATION FLOW THROUGH THE EW

A number of studies have shown that pupillomotor activity can be modulated by nonvisual factors. For example, as a subject becomes fatigued, their pupils will tend to tonically constrict and the subject will show a reduced amplitude of pupilloconstriction in response to the same light stimulus (e.g. Lowenstein and Loewenfeld, 1964). Generally, as described below, most studies have explained these tonic changes in pupil diameter and changes in pupillomotor sensitivity by assuming that there are non-visual modulatory influences on pupillomotor neurons within the EW. To my knowledge, little attention has

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been paid to possible modulatory influences on accommodation-related or choroidalrelated neurons within the EW.

Studies have shown that electrical stimulation of the posterior hypothalamus produces pupil dilatation, presumably because it elicits a “defence reaction” that impinges on neurons of the EW (e.g. Loewenfeld, 1958; Sillito and Zbrozyna, 1970a; Koss, Gherezghiher and Nomura, 1984). Similarly, electrical stimulation of the sciatic nerve elicits pupil dilatation, presumably through pain pathways that modulate EW neuron activity (e.g. Koss, Gherezghiher and Nomura, 1984). Finally, it has been shown that electrical stimulation of the medullary reticular formation or paramedian pontine reticular formation elicits pupil dilatation, presumably by activating ascending adrenergic or serotonergic pathways to the EW (Bonvallet and Zbrozyna, 1963; Loewy, Araujo and Kerr, 1973; Koss, Gherezghiher and Nomura, 1984). Consistent with these suggested inputs, adrenergic fibres have been reported in the EW of the rat (e.g. Dahlström et al., 1964), and enkephalinergic fibres have been reported in the EW of the pigeon (Gamlin and Reiner, 1987). Further, opiate receptors of both the µ- and δ-subtypes have been reported in the pigeon EW (Reiner et al. 1989). Also, pharmacological studies in rats and cats have clearly demonstrated that adrenergic inputs to the EW modulate the activity of pupil-related neurons by selectively activating α2-adrenoreceptors (Koss, Gherezghiher and Nomura, 1984; Koss, 1986; Heal et al., 1995). Furthermore, micro-injections of either clonidine, an α2-adrenoceptor agonist, or morphine, an opiate, into the EW of cats produces a well-defined pupil dilatation, presumably by inhibiting the activity of pupilrelated EW neurons (Sharpe and Pickworth, 1985). Based on these data, it appears likely that adrenergic, serotonergic, and opiate-containing pathways impinge directly on the EW and that these three neurochemically distinct pathways modulate the activity of pupilrelated EW neurons and the flow of information through this nucleus.

ACKNOWLEDGMENTS

I would like to thank Dr. Anton Reiner for his comments on this manuscript. I would like to acknowledge the support of NEI grants R01 EY-07558 and EY-09380, and of NEI CORE grant P30 EY-03039.

REFERENCES

Akert, K., Glicksman, M.A., Lang, W., Grob, P. and Huber, A. (1980). The EdingerWestphal nucleus in the monkey. A retrograde tracer study. Brain Research, 184, 491– 498.

Ariens-Kappers, C.U., Huber, G.C. and Crosby, E.C. (1936). The Comparative Anatomy of the Nervous System of Vertebrates, including Man. New York: Macmillan.

Bando, T., Ishihara, A. and Tsukahara, N. (1979). The mode of cerebellar control of lens accommodation. In Integrative Control Functions of Brain, Volume 2, edited by M.Ito, N.Tsukahara, K.Kubota and K.Yagi, pp. 149–150. Amsterdam, Oxford: Elsevier/North Holland Biomedical Press

Functions of the edinger-westphal nucleus 175

Bando, T., Tsukuda, K., Yamamoto, N., Maeda, J. and Tsukahara, N. (1984). Physiological identification of midbrain neurons related to lens accommodation in cats.

Journal of Neurophysiology, 52, 870–878.

Barbas-Henry, H.A. and Lohman, A.H. (1988). The motor nuclei and sensory neurons of the IIIrd, IVth, and VIth cranial nerves in the monitor lizard, Varanus exanthematicus. Journal of Comparative Neurology, 267, 370–386.

Bender, M.B. and Weinstein, E.A. (1943). Functional representation in the oculomotor and trochlear nuclei. Archives of Neurology and Psychiatry (Chicago), 49, 98–106.

Benevento, L.A., Rezak, M. and Santos-Anderson, R. (1977). An autoradiographic study of the projections of the pretectum in the rhesus monkey (Macaca mulatto): evidence for sensorimotor links to the thalamus and oculomotor nuclei. Brain Research 127, 197–218.

Berman, N. (1977). The connections of the pretectum in the cat. Journal of Comparative Neurology, 174, 227–254.

Bernheimer, S. (1909). Weitere experimentelle Studien zur Kenntnis der Lage des Sphincterund Levatorkerns. Albrecht von Graefes Archiv für Ophthalmologie, 70,

539–562.

Bill, A. and Nilsson, S.F. (1985). Control of ocular blood flow. Journal of Cardiovascular Pharmacology, 7, 96–102.

Bill, A. and Sperber, G.O. (1990). Control of retinal and choroidal blood flow. Eye, 4, 319–325.

Bonvallet, M. and Zbrozyna, A. (1963). Les commandes réticulaires du système autonome et en particulier de l’innervation sympathique et parasympathique de la pupille. Archives Italienne de Biologie, 101, 174–207.

Burde, R.M. and Loewy, A.D. (1980). Central origin of oculomotor parasympathetic neurons in the monkey. Brain Research, 198, 434–439.

Burde, R.M., Parelman, J.J. and Luskin, M. (1982). Lack of unity of Edinger-Westphal nucleus projections to the ciliary ganglion and spinal cord: a double-labeling approach.

Brain Research, 249, 379–382.

Burde, R.M. and Williams, F. (1989). Parasympathetic nuclei. Brain Research, 498, 371– 375.

Buttner-Ennever, J.A., Cohen, B., Horn, A.K. and Reisine, H. (1996). Pretectal projections to the oculomotor complex of the monkey and their role in eye movements.

Journal of Comparative Neurology, 366, 348–359.

Cabot, J.B., Reiner, A. and Began, N. (1982). Avian bulbospinal pathways: Anterograde and retrograde studies of cells of origin, funicular trajectories and laminar terminations.

Progress in Brain Research, 57, 79–108.

Carpenter, M.B. and Peter, P. (1970). Accessory oculomotor nuclei in the monkey.

Journal für Hirnforschung, 12, 405–418.

Carpenter, M.B. and Pierson, R.J. (1973). Pretectal region and the pupillary light reflex. An anatomical analysis in the monkey. Journal of Comparative Neurology 149, 271– 300.

Chin, N.B., Ishikawa, S., Lappin, H., Davidowitz, J. and Breinin, G.M. (1968). Accommodation in monkeys induced by midbrain stimulation. Investigative Ophthalmology, 7, 386–396.

Nervous control of the eye 176

Clarke, R.J. and Gamlin, P.D.R. (1995). Latency and dynamics of pupilloconsrriction determined by microstimulation of the Edinger-Westphal nucleus and oculomotor nerve in the primate. Society for Neuroscience Abstracts, 21, 1918.

Clarke, R.J., Coimbra, C.J. and Alessio, M.L. (1985a). Oculomotor areas involved in the parasympathetic control of accommodation and pupil size in the marmoset (Callithrix jacchus). Brazilian Journal of Medical and Biological Research, 18, 373–379.

Clarke, R.J., Coimbra, C.J. and Alessio, M.L. (1985b). Distribution of parasympathetic motoneurones in the oculomotor complex innervating the ciliary ganglion in the marmoset (Callithrix jacchus). Acta Anatomica, 121, 53–58.

Collier, J. (1927). Nuclear ophthalmoplegia, with special reference to retraction of the lids and ptosis and to lesions of the posterior commissure. Brain, 50, 488–498.

Cowan, W.M. and Wenger, E. (1968). Degeneration in the nucleus of origin of the preganglionic fibres to the chick ciliary ganglion following early removal of the optic vesicle . Journal of Experimental Zoology, 168, 105–124.

Crawford, K., Terasawa, E. and Kaufman, P.L. (1989). Reproducible stimulation of ciliary muscle contraction in the cynomologous monkey via a permanent indwelling midbrain electrode. Brain Research, 503, 265–272.

Cuthbertson, S., White, J., Fitzgerald, M.E., Shih, Y.F. and Reiner, A. (1996). Distribution within the choroid of cholinergic nerve fibers from the ciliary ganglion in pigeons. Vision Research, 36, 775–786.

Cuthbertson, S.L., Jackson, B., Toledo, C.B., Fitzgerald, M.E., Shih, Y.F., Zagvazdin, Y. and Reiner, A. (1997). Innervation of orbital and choroidal blood vessels by the pterygopalatine ganglion in pigeons. Journal of Comparative Neurology, 386, 422– 442.

Dahlström, A., Fuxe, K., Hillarp, N. and Malmfors, T. (1964). Adrenergic mechanisms in the pupillary light reflex pathway. Acta Physiologica Scandinavica, 62, 119–124.

Edinger, L. (1885). Ueber den Verlauf der centralen Hirnnervenbahnen mit Demonstration von Präparaten. Archiv für Psychiatric und Nervenkrankheim, 16, 858– 859.

Erichsen, J.T., Karten, H.J., Eldred, W.D. and Brecha, N.C. (1982). Localization of substance P-like and enkephalin-like immunoreactivity within preganglionic terminals of the avian ciliary ganglion: light and electron microscopy. Journal of Neuroscience, 2, 994–1003.

Fitzgerald, M.E., Vana, B.A. and Reiner, A. (1990a). Control of choroidal blood flow by the nucleus of Edinger-Westphal in pigeons: a laser Doppler study. Investigative Ophthalmology and Visual Science, 31, 2483–2492.

Fitzgerald, M.E., Vana, B.A. and Reiner, A. (1990b). Evidence for retinal pathology following interruption of neural regulation of choroidal blood flow: Müller cells express GFAP following lesions of the nucleus of Edinger-Westphal in pigeons. Current Eye Research, 9, 583–598.

Fitzgerald, M.E., Gamlin, P.D., Zagvazdin, Y. and Reiner, A. (1996). Central neural circuits for the light-mediated reflexive control of choroidal blood flow in the pigeon eye: a laser Doppler study. Visual Neuroscience, 13, 655–669.

Gamlin, P.D.R. and Clarke, R.J. (1995). The pupillary light reflex pathway of the primate. Journal of the American Optical Association, 66, 415–418.

Functions of the edinger-westphal nucleus 177

Gamlin, P.D.R. and Mays, L.E. (1992). Dynamic properties of medial rectus motoneurons during vergence eye movements . Journal of Neurophysiology, 67, 64– 74.

Gamlin, P.D.R. and Reiner, A. (1987). The avian Edinger-Westphal nucleus: Sources of input controlling accommodation, pupilloconstriction and choroidal blood flow.

Society for Neuroscience Abstracts, 13, 173.

Gamlin, P.D.R. and Reiner, A. (1991). The Edinger-Westphal nucleus: Sources of input influencing accommodation, pupilloconstriction and choroidal blood flow. Journal of Comparative Neurology, 306, 425–438.

Gamlin, P.D.R., Reiner, A. and Karten, H.J. (1982). Substance P-containing neurons of the avian suprachiasmatic nucleus project directly to the nucleus of Edinger-Westphal.

Proceedings of the National Academy of Sciences USA, 79, 3891–3895.

Gamlin, P.D.R., Reiner, A., Erichsen, J.T., Cohen, D.H. and Karten, H.J. (1984). The neural substrate for the pupillary light reflex in the pigeon (Columba livid). Journal of Comparative Neurology, 226, 523–543.

Gamlin, P.D.R., Zhang, Y., Clendaniel, R.A. and Mays, L.E. (1994). Behavior of identified Edinger-Westphal neurons during ocular accommodation. Journal of Neurophysiology, 72, 2368–2382.

Gamlin, P.D.R., Zhang, H. and Clarke, R.J. (1995). Luminance neurons in the pretectal olivary nucleus mediate the pupillary light reflex in the rhesus monkey. Experimental Brain Research, 106, 169–176.

Gherezghiser, T., Hey, J. and Koss, M. (1989). Cholinergic control of intraocular pressure. Investigative Ophthalmology and Visual Science (Suppl), 30, 20.

Gherezghiser, T., Hey, J.A. and Koss, M.C. (1990). Parasympathetic nervous control of intraocular pressure. Experimental Eye Research, 50, 457–462.

Gilmartin, B. (1986). A review of the role of sympathetic innervation of the ciliary muscle in ocular accommodation. Ophthalmic and Physiological Optics, 6, 23–37.

Goldor, H. and Gay, A.J. (1967). Chorioretinal vascular occlusions with latex microspheres (a long-term study). Investigative Ophthalmology, 6, 51–58.

Graybiel, A.M. and Hartwieg, E.A. (1974). Some afferent connections of the oculomotor complex in the cat: an experimental study with tracer techniques. Brain Research, 81, 543–551.

Hare, W.K., Magoun, H.W. and Ranson, S.W. (1935). Pathways for pupillary constriction: Location of synapses in the path for the pupillary light reflex and of constrictor fibers of cortical origin. Archives of Neurology and Psychiatry, 34, 1188– 1194.

Heal, D.J., Prow, M.R., Butler, S.A. and Buckett, W.R. (1995). Mediation of mydriasis in conscious rats by central postsynaptic α2-adrenoceptors. Pharmacology, Biochemistry and Behavior, 50, 219–224.

Helmholtz, H. (1867). Handbuch der Physiologischen Optik. Leipzeig, Voss.

Hosoba, M., Bando, T. and Tsukahara, N. (1978). The cerebellar control of accommodation of the eye in the cat. Brain Research, 153, 495–505.

Hultborn, H., Mori, K. and Tsukahara, N. (1973). The neuronal pathway subserving the pupillary light reflex and its facilitation from cerebellar nuclei. Brain Research, 63, 357–361.

Nervous control of the eye 178

Hultborn, H., Mori, K. and Tsukahara, N. (1978). Cerebellar influence on parasympathetic neurones innervating intra-ocular muscles. Brain Research, 159, 269– 278.

Inoue, T. (1980). The response of rabbit ciliary nerve to luminance intensity. Brain Research, 201, 206–209.

Ijichi, J., Kiyohara, T., Hosoba, M. and Tsukahara, N. (1977). The cerebellar control of the pupillary light reflex. Brain Research, 128, 69–79.

Ishikawa, S., Sekiya, H. and Kondo, Y. (1990). The center for controlling the near reflex in the midbrain of the monkey: a double labelling study. Brain Research, 519, 217– 222.

Jaeger, R.J. and Benevento, L.A. (1980). A horseradish peroxidase study of the innervation of the internal structures of the eye. Investigative Ophthalmology and Visual Science 19, 575–583.

Jampel, R.S. and Mindel, J. (1967). The nucleus for accommodation in the midbrain of the macaque. The effect of accommodation, pupillary constriction, and extraocular muscle contraction produced by stimulation of the oculomotor nucleus on the intraocular pressure. Investigative Ophthalmology, 6, 40–50.

Johnson, DA. and Purves, D. (1981). Post-natal reduction of neural unit size in the rabbit ciliary ganglion. Journal of Physiology, 318, 143–159.

Johnson, D.A. and Purves, D. (1983). Tonic and reflex synaptic activity recorded in ciliary ganglion cells of anaesthetized rabbits. Journal of Physiology, 339, 599–613.

Judge S.J. and Cumming B.G. (1986). Neurons in the monkey midbrain with activity related to vergence eye movement and accommodation. Journal of Neurophysiology, 55, 915–930.

Karplus, J.P. and Kreidl, A. (1913). Ueber die Bahn des Pupillarreflexes. Pflugers Archiv für die Gestame Physiologie de Menschen und der Tiere, 149, 115–155.

Keane, J.R. and Davis, R.L. (1976). Pretectal syndrome with metastatic malignant melanoma to the posterior commissure. American Journal of Ophthalmology, 82, 910– 914.

Koss, M.C., Gherezghiher, T. and Nomura, A. (1984). CNS adrenergic inhibition of parasympathetic oculomotor tone. Journal of the Autonomic Nervous System, 10, 55– 68.

Koss, M.C. (1986). Pupillary dilation as an index of central nervous system alpha 2- adrenoceptor activation. Journal of Pharmacological Methods, 15, 1–19.

Kourouyan, H and Horton, J.C. (1997). Transneuronal retinal input to the primate Edinger-Westphal nucleus. Journal of Comparative Neurology, 381, 68–80.

Kruger, P. (1979). Infrared recording retinoscope for monitoring accommodation.

American Journal of Optometry and Physiological Optics, 56, 116–123.

Kuchiiwa, S., Kuchiiwa, T. and Suzuki, T. (1989). Comparative anatomy of the accessory ciliary ganglion in mammals. Anatomy and Embryology, 180, 199–205.

Kuchiiwa, S., Kuchiiwa, T. and Nakagawa, S. (1994). Localization of preganglionic neurons of the accessory ciliary ganglion in the midbrain: HRP and WGA-HRP studies in the cat. Journal of Comparative Neurology, 340, 577–591.

Kuhlenbeck, H. (1937). The ontogenetic development of the diencephalic centers in the bird’s brain (chicken) and comparison with the reptilian and mammalian diencephalon.

Functions of the edinger-westphal nucleus 179

Journal of Comparative Neurology, 66, 23–75.

Kuhlenbeck, H. (1939). The development and structure of the pretectal cell masses in the chick. Journal of Comparative Neurology, 71, 361–387.

Lindsberg, P.J., O’Neill, J.T., Paakkari, I., Hallenbeck, J. and Feuerstein, G. (1989). Validation of laser-doppler flowmetry in measurements of spinal cord blood flow.

American Journal of Physiology, 257, 674–680.

Lindvall, O., Bjorklund, A., Nobin, A. and Stenevi, A. (1974). The adrenergic innervation of the rat thalamus as revealed by the glyoxylic acid fluorescence method.

Journal of Comparative Neurology, 154, 317–348.

Ljungdahl, A., Hökfelt, T. and Nilsson, G. (1978). Distribution of Substance P-like immunoreactivity in the central nervous system of the rat–I. Cell bodies and nerve terminals. Neuroscience, 3, 861–943.

Loewenfeld, I.E. (1958). Mechanisms of reflex dilatation of the pupil. Historical review and experimental analysis. Documenta Ophthalmologica, 12, 185–448.

Loewenfeld, I.E. (1993). The Pupil: Anatomy Physiology, and Clinical Applications.

Ames: Iowa State University Press.

Loewy, A.D., Araujo, J.C. and Kerr, F.W.L. (1973). Pupillodilator pathways in the brain stem of the cat: Anatomical and electrophysiological identification of a central autonomic pathway. Brain Research, 60, 65–91.

Loewy, A.D. and Saper, C.B. (1978). Edinger-Westphal nucleus: projections to the brainstem and spinal cord in the cat. Brain Research, 150, 1–27.

Loewy, A.D., Saper, C.B. and Yamodis, N.D. (1978). Re-evaluation of the efferent projections of the Edinger-Westphal nucleus in the cat. Brain Research, 141, 153–159.

Lowenstein, O. (1954). Clinical pupillary symptoms in lesions of the optic nerve, optic chiasm and optic tract. Archives of Ophthalmology, 52, 385–403.

Lowenstein, O. and Loewenfeld, I.E. (1964). The sleep-waking cycle and pupillary activity. Annals of the New York Academy of Sciences, 117, 142–156.

Lowenstein, O. and Loewenfeld, I.E. (1969). The Pupil. In The Eye, Vol. 3, edited by H.Davson, pp. 255–337. New York: Academic Press.

Lyman, D. and Mugnaini, E. (1980). The avian accessory oculomotor nucleus. Society for Neuroscience Abstracts, 6, 479.

Magnuson, D.J., Rezak, M. and Benevento, L.A. (1980). Some afferent connections to the oculomotor complex in the macaque monkey. Society for Neuroscience Abstracts, 6, 478.

Magoun, H.W. and Ranson, S.W. (1935a). The central path of the light reflex: A study of the effect of lesions. Archives of Ophthalmology, 13, 791–811.

Magoun, H.W. and Ranson, S.W. (1935b). The afferent path of the light reflex: A review of the literature. Archives of Ophthalmology, 13, 862–874.

Magoun, H.W., Atlas, D., Hare, W.K. and Ranson, S.W. (1936). The afferent path of the pupillary light reflex in the monkey. Brain 59, 234–249.

Marg, E., Reeves, J.L. and Wendt, W.E. (1954). Accommodative response of the eye to electrical stimulation of the ciliary ganglion in cats. American Journal of Optometry, 31, 127–136.

Marwitt, R., Pilar, G. and Weakly, J.N. (1971). Characterization of two ganglion cell populations in avian ciliary ganglion. Brain Research, 25, 317–334.

Nervous control of the eye 180

Mays, L.E. (1984). Neural control of vergence eye movements: convergence and divergence neurons in the midbrain. Journal of Neurophysiology, 51, 1091–1108.

May, P.J. and Warren, S. (1993). Ultrastructure of the macaque ciliary ganglion. Journal of Neurocytology, 22, 1073–1095.

Mays L.E., Porter J.D., Gamlin P.D.R and Tello C.A. (1986). Neural control of vergence eye movements: neurons encoding vergence velocity. Journal of Neurophysiology, 56, 1007–1021.

May, P.J., Porter, J.D. and Gamlin, P.D.R. (1992). Interconnections between the primate cerebellum and midbrain near-response regions Journal of Comparative Neurology, 315, 98–116.

MeInitchenko, L.V. and Skok, V.I. (1970). Natural electrical activity in mammalian parasympathetic ganglion neurons. Brain Research, 23, 277–279.

Meriney, S.D. and Pilar, G.J. (1987). Cholinergic innervation of the smooth muscle cells in the choroid coat of the chick eye and its development. Journal of Neuroscience, 7, 3827–3839.

Nakanome, Y., Karita, K., Izumi, H. and Tamai, M. (1995). Two types of vasodilation in cat choroid elicited by electrical stimulation of the short ciliary nerve. Experimental Eye Research, 60, 37–42.

Narayanan, C.H. and Narayanan, Y. (1976). An experimental inquiry into the central source of preganglionic fibers to the chick ciliary ganglion. Journal of Comparative Neurology, 166, 101–110.

Nicholson, J. and Severin, C.M. (1981). Afferent projections of the Edinger-Westphal nucleus in the rat. Anatomical Record, 199, 183.

Nisida, I. and Okada, H. (1960). The activity of the pupilloconstrictory centers. Japanese Journal of Physiology, 10, 64–72.

Olmsted, J.M.D. (1944). The role of the autonomic nervous system in accommodation for far and near vision. Journal of Nervous and Mental Disease, 99, 794–798.

Parelman, J.J., Fay, M.T. and Burde, R.M. (1984). Confirmatory evidence for a direct parasympathetic pathway to internal eye structures. Transactions of the American Ophthalmological. Society, 82, 371–380.

Parver, L.M., Auker, C.R., Carpenter, D.O. and Doyle, T. (1982). Choroidal blood flow II. Reflexive control in the monkey. Archives of Ophthalmology, 100, 1327–1330.

Parver, L.M., Auker, C.R. and Carpenter, D.O. (1983). Choroidal blood flow. III. Reflexive control in human eyes. Archives of Ophthalmology, 101, 1604–1606.

Pasik, P., Pasik, T. and Bender, M.B. (1969). Autonomic dysfunctions in monkeys with pretectal lesions. Federation Proceedings, 28, 3175.

Pierson, R.J. and Carpenter, M.B. (1974). Anatomical analysis of pupillary reflex pathways in the Rhesus monkey. Journal of Comparative Neurology, 158, 121–144.

Pilar, G. and Turtle, J.B. (1982). A simple neuronal system with a range of uses: The avian ciliary ganglion. In Progress in Cholinergic Biology: Model Cholinergic synapses, edited by A.Goldberg and I.Hanin, pp. 213–247. New York: Raven Press.

Pitts, D.G. (1967). Electrical stimulation of oculomotor nucleus: The effects of stimulus voltage and anesthesia. American Journal of Optometry, 44, 505–516.

Ponsford, J.R., Bannister, R. and Paul, E.A. (1982). Methacholine pupillary responses in third nerve palsy and Adie’s syndrome. Brain, 105, 583–597.

Functions of the edinger-westphal nucleus 181

Powers, A.S. and Reiner, A. (1993). The distribution of Cholinergic neurons in the central nervous system of the turtle. Brain, Behavior and Evolution, 41, 326–345.

Ranson, S.W. and H.W. Magoun (1933). The central path of the pupilloconstrictor reflex in response to light. Archives of Neurology and Psychiatry (Chicago), 30, 1193–1204.

Reiner, A., Karten, H.J., Gamlin, P.D.R. and Erichsen, J.T. (1983). Parasympathetic ocular control: Functional subdivisions and circuitry of the avian nucleus of EdingerWestphal. Trends in Neurosciences, 6, 140–145.

Reiner, A., Brauth, S.E., Kitt, C.A. and Quirion, R. (1989). Distribution of µ, δ, and κ opiate receptor types in the forebrain and midbrain of pigeons. Journal of Comparative Neurology, 280, 359–382,

Reiner, A., Erichsen, J.T., Cabot, J.B., Evinger, C., Fitzgerald, M.E. and Karten, H.J. (1991). Neurotransmitter organization of the nucleus of Edinger-Westphal and its projection to the avian ciliary ganglion. Visual Neuroscience, 6, 451–472.

Ripps, H., Breinin, G.M. and Baum, J.L. (1961). Accommodation in the cat. Transactions of the American Ophthalmological Society, 59, 176–193.

Roste, G.K. and Dietrichs, E. (1988). Cerebellar cortical and nuclear afferents from the Edinger-Westphal nucleus in the cat. Anatomy and Embryology, 178, 59–65.

Ruskell, G.L. (1971). Facial parasympathetic innervation of the choroidal blood vessels in monkeys. Experimental Eye Research, 12, 166–172.

Ruskell, G.L. (1990). Accommodation and the nerve pathway to the ciliary muscle: a review. Ophthalmological and Physiological Optics, 10, 239–242.

Ruskell, G.L. and Griffiths, T. (1979). Peripheral nerve pathway to the ciliary muscle.

Experimental Eye Research, 28, 277–284.

Scalia, F. (1972). The termination of retinal axons in the pretectal region of mammals.

Journal of Comparative Neurology, 145, 223–257.

Schaeffel, F., Troilo, D., Wallman, J. and Rowland, H.C. (1990). Developing eyes that lack accommodation grow to compensate for imposed defocus. Visual Neuroscience, 4, 177–183.

Scherer, S.S. (1986). Reinnervation of the extraocular muscles in goldfish is nonselective.

Journal of Neuroscience, 6, 764–773.

Schlag, J.D. and Fuller, J.H. (1976). Determination of antidromic excitation by the collision test: problems of interpretation. Brain Research, 112, 283–298.

Sekiya, H., Kawamura, K. and Ishikawa, S. (1984). Projections from the EdingerWestphal complex of monkeys as studied by means of retrograde axonal transport of horseradish peroxidase. Archives Italienne de Biologic, 122, 311–319.

Sharpe, L.G. and Pickworth, W.B. (1985). Opposite pupillary size effects in the cat and dog after microinjections of morphine, normorphine and clonidine in the EdingerWestphal nucleus. Brain Research Bulletin, 15, 329–333.

Shih, Y.F., Fitzgerald, M.E. and Reiner, A. (1993). Effect of choroidal and ciliary nerve transection on choroidal blood flow, retinal health, and ocular enlargement. Visual Neuroscience, 10, 969–979.

Sillito, A.M. and Zbrozyna, A.W. (1970a). The activity characteristics of the preganglionic pupilloconstrictor neurones. Journal of Physiology (London), 211, 767– 779.

Sillito, A.M. and Zbrozyna, A.W. (1970b). The localization of pupilloconstrictor function

Nervous control of the eye 182

within the midbrain of the cat. Journal of Physiology (London), 211, 461–477.

Steiger, H-J. and Büttner-Ennever, J.A. (1979). Oculomotor nucleus afferents in the monkey demonstrated with horseradish peroxidase. Brain Research, 160, 1–15.

Stjernschantz, J., Alm, A. and Bill, A. (1976). Effects of intracranial oculomotor nerve stimulation on ocular blood flow in rabbits: Modification by indomethacin.

Experimental Eye Research, 23, 461–469.

Sugimoto, T., Itoh, K. and Mizuno, N. (1977). Localization of neurons giving rise to the oculomotor parasympathetic outflow: A HRP study in cat. Neuroscience Letters, 7, 301–305.

Sugimoto, T., Itoh, K. and Mizuno, N. (1978). Direct projections from the EdingerWestphal nucleus to the cerebellum and spinal cord in the cat: An HRP study.

Neuroscience Letters, 9, 17–22.

Sun, W. and May, P.J. (1993). Organization of the extraocular and preganglionic motoneurons supplying the orbit in the lesser Galago. Anatomical Record, 237, 89– 103.

Thomas, D.M., Kaufman, R.P., Sprague, J.M. and Chambers, W.W. (1956). Experimental studies of the vermal cerebellar projections in the brain stem of the cat (Fastigiobulbar tract). Journal of Anatomy, 90, 371–385.

Thompson, H.S. (1987). The Pupil. In Adler’s Physiology of the Eye: Clinical Application, 8th edn., edited by R.A.Moses and W.M.Hart, Jr., St. Louis: C.V.Mosby Co.

Tornqvist, G. (1966). Effect of cervical sympathetic stimulation on accommodation in monkeys. Acta Physiologica Scandinavica, 67, 363–372.

Tornqvist, G. (1967). The relative importance of the parasympathetic and sympathetic nervous systems for accommodation in monkeys. Investigative Ophthalmology, 6, 612–617.

Toyoshima, K., Kawana, E. and Sakai, H. (1980). On the neuronal origin of the afferents to the ciliary ganglion in cat. Brain Research, 185, 67–76.

Troilo, D. and Wallman, J. (1987). Changes in corneal curvature during accommodation in chicks. Vision Research, 27, 241–247.

Tsukahara, N., Kiyohara, T. and Ijichi, Y. (1973). The mode of cerebellar control of pupillary light reflex. Brain Research, 60, 244–248.

Warwick, R. (1954). The ocular parasympathetic nerve supply and its mesencephalic sources. Journal of Anatomy, 88, 71–93.

Wathey, J.C. (1988). Identification of the teleost Edinger-Westphal nucleus by retrograde horseradish peroxidase labeling and by electrophysiological criteria. Journal of Comparative Physiology A, 162, 511–524.

Wathey, J.C. and Wullimann, M.F. (1988). A double-label study of efferent projections from the Edinger-Westphal nucleus in goldfish and kelp bass. Neuroscience Letters, 93, 121–126.

Weber, J.T and Halting, J.K. (1980). The efferent projections of the pretectal complex: An autoradiographic and horseradish peroxidase analysis. Brain Research, 194, 1–28.

Westheimer, G. and Blair, S.M. (1973). The parasympathetic pathways to internal eye muscles. Investigative Ophthalmology, 12, 193–197.

Westphal, C. (1887). Ueber einen Fall von chronischer progressiver Lähmung der

Functions of the edinger-westphal nucleus 183

Augenmuskeln (ophthalmoplegia externa) nebst Beschreibung von Ganglienzellengruppen in Bereiche des Oculomotoriuskerns. Archiv für Psychiatric und Nervenkrankheim, 18, 846–871.

Young, M.J. and Lund, R.D. (1994). The anatomical substrates subserving the pupillary light reflex in rats: origin of the consensual pupillary response. Neuroscience, 62, 481– 496.

Zhang, H.Y. and Gamlin, P.D.R. (1996). Single-unit activity within the posterior fastigial nucleus during vergence and accommodation in the alert primate. Society for Neuroscience Abstracts, 22, 2034.

Zhang, Y., Gamlin, P.D.R. and Mays, L.E. (1991). Antidromic identification of midbrain near response cells projecting to the oculomotor nucleus. Experimental Brain Research, 84, 525–528.

Zhang, Y., Mays, L.E. and Gamlin, P.D.R. (1992). Characteristics of near response cells projecting to the oculomotor nucleus. Journal of Neurophysiology, 67, 944–960.

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INTRODUCTION

The regulation of blood flow to the retina is a critical factor in the maintenance of normal retinal function and any failure of the processes regulating retinal perfusion is likely to have a profound effect on retinal homeostasis. In particular, dysfunctional retinal blood flow has been associated with a variety of retinal disorders including glaucoma and hypertensive retinopathy. The retina receives its blood supply from two separate and distinct vascular beds. In general, the choroidal vasculature supplies the outer, avascular region of the retina (the photoreceptor layer) by diffusion across the retinal pigmented epithelium (RPE). The retinal vasculature is normally confined to the inner retina as far as the boundary between the inner nuclear layer and outer plexiform layer, and supplies these inner layers as well as 10% of the oxygen requirements of the photoreceptors during dark adaptation (Ahmed et al., 1993).

The vessels comprising these two anatomically discrete vasculatures differ extensively in their phenotype (Greenwood, 1992; Greenwood et al., 1995) as well as in the level of autonomic innervation and autoregulation of blood flow within them (Bill and Nilsson, 1985; Bill and Sperber, 1990; Haefliger et al., 1994; Brown and Jampol, 1996; Funk, 1997). It is generally believed that blood flow to the choroid is regulated predominantly through autonomic activity, whilst that to the retina is almost entirely through autoregulation. Despite the importance of the retinal blood supply the precise mechanisms controlling retinal perfusion remain poorly understood. Recently, however, evidence is emerging that retinal vascular tone may also be controlled to some degree by direct intrinsic innervation. Regulation of retinal blood flow, including both vascular innervation and autoregulatory mechanisms, should not be considered in isolation but must be examined in the context of the unique nature and importance of the retinal vasculature and the considerable retinal metabolic demands. In this review we will summarise the current understanding of the control of blood flow to the retina and speculate on the possible involvement of retinal vascular innervation in controlling this process.

BLOOD SUPPLY TO THE RETINA

Before examining the factors controlling retinal perfusion, it is important that the organisation of the blood supply to the retina should be understood. The retina is supplied by the central retinal artery where, in the human, it arises from the ophthalmic artery approximately 1 cm behind the eye. With the central retinal vein it enters the optic nerve and passes forward in the centre of the optic nerve, through a gap in the lamina cribrosa and emerges centrally through the papilla. As described in more detail below, this region forms an important point of demarcation between the different predominant factors controlling flow through the central retinal artery and its branches and that of the retinal vasculature. From here, the central retinal artery branches into the superior and inferior branches which then subdivide into nasal and temporal arteries. The superior and inferior

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temporal arteries curve above and below the macula and foveal regions. In approximately 20% of people there exists a cilioretinal artery (arising from a posterior ciliary artery) which forms a small anastomotic connection between the choroidal and retinal circulation and explains why macula function may be preserved in central retinal artery occlusion. At present, it is unclear whether the innervation of the branches of the posterior ciliary artery that form these connections extend into the retina itself or whether, like those of the central retinal artery, they stop at the point of entry (Ye, Laties and Stone, 1990).

Each of the large retinal arterial branches projects through the nerve fibre layer beneath the inner limiting membrane. There are four major branches each supplying a quadrant of the retina and, as no overlap exists between them, these vessels are considered to be functional end-arteries. Throughout the retina there exist two main levels of capillary networks (although this pattern does vary in some areas). The inner plexus, which is situated at the level of the ganglion cell layer, and the outer plexus, which is at the level of the deep aspect of the inner nuclear layer. The outer retina (outer plexiform and photoreceptor layers) and a 500 µm diameter region centred on the fovea are avascular. The density of capillaries varies, being most dense at the macula, decreasing in density towards the peripheral retina where neuronal populations are also less dense. Capillary free zones also surround arterioles and arteries.

The outer retina and the entire thickness of the foveal retina derive much of their nutrition from the choroidal circulation, which lies beneath the RPE extending from the optic nerve margins to the periphery of the retina where it is continuous with the vascular plexus of the ciliary body. This vascular bed is supplied predominantly by the two long posterior ciliary arteries and the short posterior ciliary arteries, with some contribution arising from anastomoses with the anterior ciliary arteries. The capillary bed of the choroidal circulation is the choriocapillaris, a wide-bore (20–40 µm), fenestrated capillary network which abuts Bruch’s membrane, a basement membrane-like layer which inter-venes between the choriocapillaris and the RPE.

The primate choroid, unlike the retinal vasculature, is richly innervated, particularly at the posterior pole, in the vicinity of the fovea (Flugel et al., 1994; Flugel-Koch, Kaufman and Lutjen-Drecoll, 1994). Evidence supports the involvement of parasympathetic fibres from the pterygopalatine ganglion (Ruskell, 1971) and sympathetic innervation from the superior cervical ganglion (Nuzzi, Gugleilmone and Grignolo, 1995; Klooster et al., 1996). Stimulation of sympathetic nerves from the cervical sympathetic ganglion brings about a frequency-dependent vasoconstriction of choroidal vessels and is thought to prevent choroidal overperfusion (Bill and Sperber, 1990). Conversely, stimulation of the parasympathetic facial nerve results in increased blood flow (Stjernschantz and Bill, 1980).

THE BLOOD-RETINAL BARRIER

The retinal vascular endothelium is highly specialised, forming the inner blood-retinal barrier (BRB), the outer BRB being formed by the RPE. This selective cellular barrier regulates the passage of molecules and cells, through specific receptors and nutrient transport systems, both into and out of the retina, helping to maintain homeostasis. The endothelial cells of the retinal vasculature are identical in most respects to the endothelia

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that form the blood-brain barrier. The morphological correlates of this vascular barrier are the tight junctions between endothelial cells which are capable of excluding ions, as well as a lack of pores, fenestrations and vesicular activity. In addition to this physical barrier, both brain and retinal endothelium express a unique repertoire of surface receptors and may thus respond to external signals differently from those of non-CNS endothelium. It is important to consider, therefore, that the release of vasoactive factors involved in the regulation of local blood flow, as described below, may also affect other functions of the vascular endothelium in particular its capacity to maintain a patent barrier to circulating molecules.

Retinal endothelial cells produce a thick basal lamina within which there also exist numerous pericytes, these being more abundant than in the brain. These pericytes may have important properties relating to the vascular tone, especially in the capillary bed, as they have been shown to possess contractile properties (Kelley et al., 1987; Hirschi and D’Amore, 1996). Surrounding the basement membrane of the retinal vessels is the glia limitans, comprising the processes of both astrocytes and microglia (Stone and Dreher, 1987; Provis et al., 1995). Astrocytes produce vasoactive substances and are also thought to produce factors which induce the tight, barrier phenotype within the vascular endothelium (Janzer and Raff, 1987; Raub, Luentzel and Sawada, 1992). The inclusion of microglia in the glial limitans similarly may suggest that microglia have a role in regulating barrier function (Diaz, Penfold and Provis, 1998).

In contrast to retinal vessels, the choriocapillaris is composed of permeable, fenestrated endothelia (particularly where they abut Bruch’s membrane) which do not posses any barrier properties. Between this leaky vascular bed and the neural retina lies the posterior barrier formed by a monolayer of RPE joined together by tight apical junctions, providing a selectively permeable barrier between the choroid and the neurosensory retina.

RETINAL BLOOD FLOW

The retina is extremely metabolically active, having the highest oxygen consumption per weight of any human tissue. Both the choroidal circulation (outer third) and the retinal circulation (inner two thirds) supply the retina with its metabolic requirements. Ninetyeight per cent of blood to the eye passes through the uveal tract (choroid, ciliary body and iris), of which 85% is through the choroid. The choroidal circulation has a high flow rate (150 mm/s), low oxygen extraction (only 5–10% of its oxygen being extracted) and may also provide thermoregulation. The retina on the other hand receives approximately 5% of the total blood flow to the eye and, unlike the choroid, has a low flow rate (25 mm/s) and high oxygen exchange. It has been suggested that such differences are due to the spatial relationship of the respective vascular beds to the retina. Thus, the distance for nutrients to reach the outer retina from the choroid is relatively large and requires a high concentration gradient to overcome these distances and to drive the carrier-facilitated diffusion transport processes at the posterior BRB. In particular, in order to meet the metabolic requirements of photoreceptors, oxygen must diffuse across the entire thickness of the RPE plus the length of the outer segments and inner segments (around 100 µm at the fovea) to reach mitochondria concentrated in the ellipsoid. On the other hand the retinal circulation has only to supply the parenchymal cells in their immediate

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vicinity and as such does not require such high blood-to-tissue concentration gradients to be maintained.

An important characteristic of the retinal vasculature is its phenomenal capacity to maintain constant retinal blood flow in the face of considerable alterations in both perfusion pressure and intraocular pressure (Bill and Nilsson, 1985). The mechanisms responsible for this exquisite control of blood flow to the retina are not entirely clear although it is recognised that autoregulatory processes are the primary mechanism behind the control of retinal vascular tone.

AUTOREGULATION

Since retinal vessels have no conventional sympathetic innervation (Ye, Laties and Stone, 1990), blood flow in response to raised blood pressure is largely dependent upon autoregulation. In healthy humans and experimental animals retinal blood flow is exceptionally adept at autoregulating flow in response to changes in pressure and metabolic demand. This remarkable regulatory capacity is thought to be achieved through the local vascular response to released vasoactive metabolites including eicosanoids, vasogenic amines, autocoids, peptides and nitric oxide (NO) as well as oxygen and carbon dioxide tensions, pH, intravascular pressure (myogenic response) and sheer stress. The variability of the source and site of action further compound the potential complexity of response brought about by such a diverse array of stimuli. Thus, vasoactive agents may be blood-borne or released from vascular and perivascular cells, including local intrinsic neurones, and may act either directly or indirectly, upon smooth muscle or pericytes and endothelium. It is likely that the complex interaction of these vasoactive metabolites results in a balance between vasoconstriction and vasodilatation and that vessel tone depends on their relative actions. Unlike the retina, the choroid is believed to be largely devoid of autoregulatory mechanisms partly because the supply is too far removed from the local metabolic triggers. In addition, any substances released by the local tissue which may initiate autoregulatory responses would have to overcome the outer BRB to exert their effects upon the choriocapillaris.

Many of the vasoactive metabolites reported to be involved in autoregulation are also inflammatory agents and have a variety of additional actions upon vascular endothelium including the induction of inflammatory molecules and vascular leakage. This can be illustrated by the purine vasodilator, adenosine, which has recently has been proposed as an important mediator in controlling retinal arterial tone (Braunagel, Xiao and Chiou, 1988; Gidday and Park, 1993; Crosson, DeBenedetto and Gidday, 1994). However, as with many vasoactive metabolites it may also lead to breakdown of the blood-retinal barrier and haemorrhage (Sen and Campochiaro, 1989; Campochiaro and Sen, 1989). The literature relating to autoregulation of retinal vascular blood flow is outside the main scope of this review but has been explored in more detail in a number of recent reviews (Haefliger et al., 1994; Brown and Jampol, 1996; Funk, 1997)

Evidence for the intrinsic innervation of retinal vessels 189

INNERVATION OF RETINAL VASCULATURE

Although the retinal vasculature lacks any conventional autonomic innervation, there is growing evidence for the existence of central innervation of retinal vessels which may influence retinal blood flow. Indeed, some of the vasoactive metabolites described above, can be generated by neurones and act as classical neurotransmitters. In addition, it has been recognised for many years that there is autonomic innervation of the central retinal artery and that, as the major supplier of blood flow to the retinal vasculature, such vasomotor control can influence retinal perfusion.

THE CENTRAL RETINAL ARTERY

As a branch of the internal carotid, the central retinal artery is innervated by both sympathetic and parasympathetic neurones as far as the lamina cribrosa. Beyond this point however, the presence of peripheral innervation of the vasculature has not been found to any meaningful degree. Using a histofluorometric technique to visualise catecholamines, Laties (1967) demonstrated in new world monkeys that the central retinal artery is innervated with a fine and plentiful plexus of adrenergic nerve fibres, which were also found to be present in abundance in the choroidal vessels of the same animals. Adrenergic innervation of the central retinal vein was also observed, although to a lesser extent than the artery. However, within the globe the arteriolar branches of the central retinal artery lose their adrenergic innervation although it was reported that in some animals there remained some evidence within the region of the optic disc. Beyond this point the bifurcating arterioles and capillary bed is also devoid of adrenergic innervation whilst the small branching arterioles behind the lamina cribrosa retain their innervation. This situation is slightly different from that in the brain where the plexus of adrenergic nerve fibres of the great vessels continues, albeit very much reduced, over the parenchymal arteries (Owman, Edvinsson and Nielsen, 1974).

In the rabbit, substance P (SP)-like immunoreactivity has also been detected in the central retinal artery, but not in the central retinal vein or retinal vessels (Kumagai et al., 1988) leading to the proposal that the central retinal artery is innervated by peripheral nerves whereas the vessels of the retina are innervated by the central nervous system. There is also good evidence for peptidergic innervation of the central retinal artery of the rat and monkey (Ye, Laties and Stone, 1990) where beaded nerve fibres immunoreactive to the vasoactive peptides, calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), SP and vasoactive intestinal peptide (VIP) were found to be present in the perivascular space. Both CGRP and SP immunoreactivity co-localised to the same nerve fibres. More recently, both aminergic and cholinergic vasomotor innervation of the human central retinal artery and vein have also been identified (Komai et al., 1995). Similarly, immunohistochemical identification of nitric oxide synthase immunoreactivity (NOS-IR) in nerve fibres within the adventitia and media of the central retinal artery of the dog has been observed (Toda, Kitamura and Okamura., 1994) suggesting that NO liberated from these potential vasodilator nerves may be capable of altering arterial

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muscle tone and blood flow to the retina. Thus, in addition to a role in autoregulation through local endothelial cell synthesis, NO may also be released from neurones.

The strong evidence for innervation of the central retinal artery would suggest that autonomic innervation provides a degree of control over perfusion of the retina. The relative importance of this control in the overall regulation of retinal blood flow, however, is not entirely clear.

EVIDENCE FOR RETINAL VASCULAR INNERVATION

Although retinal arteries appear to lack extrinsic innervation, the presence of autonomic receptors on their walls implies the existence of direct innervation by retinal neurones. There appear to be two candidate substances for neuromodulation of the retinal vasculature; the potent vasoactive metabolite/neurotransmitter NO, which has been strongly implicated in retinal vasodilatation (Nilsson, 1996), and SP, which is implicated in the regulation of cerebral blood flow (Edvinsson, 1985). While it is likely that alterations in NO production by the retinal vascular endothelium or other perivascular cells (Donati et al., 1995) regulates retinal blood flow, it has also been suggested that endothelial NOmediated dilatation may also be activated by SP (Kitamura et al., 1993).

Nitroxidergic processes derived from intrinsic retinal cells have been described in human and rat retina (Roufail, Stringer and Rees, 1995; Penfold and Provis, 1998) and are also present in cat retina (Provis and Penfold, unpublished observation). In the human retina, three classes of NADPH-diaphorase positive cells have been described previously, based on soma diameter and dendritic stratification and named ND1–3 (Provis and Mitrofanis, 1990). For convenience we refer to nitroxidergic cells with processes contacting the retinal vasculature, as NDv cells (Figure 5.1). NDv cells are relatively large and characteristically have an intensely positive, coarse process arising from the aspect of the cell body adjacent to a retinal vessel. Additional processes radiate from the soma and presumably contact other retinal cells. The processes of NDv cells are relatively short, reaching vessels within 50 µm, or so. At the vascular basement membrane these processes appear to become broader and flatter, as well as more intensely positive, at the point of contact. Ultrastructurally there appear to be two sizes of NOS-IR fibres associated with the basement membrane of the retinal vessels in humans (Figure 5.2). Whether these correspond to terminals of the ‘coarse’ and ‘fine’ fibres is not clear. We have seen the large NOS-IR processes opposed to the glia limitans (Figure 5.2A) while the small NOS-IR processes have been observed within the perivascular space of the retinal vessels (Figure 5.2B).

Given the important role NO plays in the relaxation of blood vessels in other parts of the body as well as, it appears, in the choroid (Flugel et al., 1994; Flugel-Koch, Kaufman and Lutjen-Drecoll, 1994) it seems likely that NOS-IR neuronal processes associated with the retinal vessels are involved in modulation of retinal blood flow, although the mechanism of this involvement is not known.

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(see B-D). (B) The two NDv cells to the right of the field in (A) at higher magnification, showing the expansions of their processes at presumed points of contact with the retinal vessel. The same cells in different planes of focus, at the soma (C) and level of vascular associations (D). Note also the patches of NADPHdiaphorase reactivity scattered along the vascular wall (A-D). (B-D) are at the same magnification.

Some of the strongest evidence to emerge for retinal vascular innervation has come from studies investigating the distribution of SP-immunoreactive (SP-IR) retinal neurones and their association with retinal vessels. Irnmunoreactiviry of retinal neurons for SP has been investigated in a range of vertebrates (for a review see Kolb et al., 1995) and several studies have reported SP-IR in neurons of the ganglion cell (GCL) and inner nuclear layers (INL) of the primate retina (Brecha et al., 1982; Marshak, 1989; Li and Lam, 1990; Provis, Yip and Penfold, 1992; Cuenca, De Juan and Kolb, 1995; Cuenca and Kolb, 1998; Penfold and Provis, 1998). In general, SP is localised in amacrine cell populations and in some species, including primates, is also contained in a population of ganglion cells (Kolb et al., 1995; Cuenca, De Juan and Kolb, 1995; Cuenca and Kolb, 1998). Since the retina has abundant amacrine-derived nerve processes containing neurotransmitters and/or neuropeptides, a local mechanism coupled to retinal activity might contribute to the local regulation of retinal blood flow. Indeed, in those few studies in which this possibility has been investigated, the presence of varicose, SP-IR, dendritic terminals associated with the retinal vasculature in primate retinae has been noted (Verstappen et al., 1986; Provis, Yip and Penfold, 1992; Cuenca, De Juan and Kolb, 1995), although their precise structural features and functional significance remain to be determined.

For sources detailing the morphology and stratification of SP immunoreactive cells and processes in human retina the reader is referred to Cuenca et al., (Cuenca, De Juan and Kolb, 1995; Cuenca and Kolb, 1998). However, two of us (JMP and PLP) have been particularly interested in defining the features of the interaction between SP-IR cell processes and the retinal vasculature.

In whole mounts of human retina the superficial aspect of the major retinal vessels is enmeshed in a network of SP-IR nerve fibres, many of which terminate in clusters of SPIR varicosities, particularly at the branch points of large vessels (Figure 5.3). In only a few circumstances was it possible to identify the cell somas from which these fibres originate (Figure 5.4) Using double immunolabelling for SP and glial fibrillary acidic protein (GFAP) to label astrocytes, these varicosites are evidently integrated with the glia limitans of the retinal vessels (Figure 5.5). Ultrastructural localisation of SP confirmedthat immunoreactive varicosites do not terminate directly on the vascular endothelium or contractile elements, but are integral to the glia limitans and are separated from the vascular endothelium by the perivascular space (Figure 5.6).

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Figure 5.2 Electron micrographs showing nitric oxide synthaseimmunoreactive (NOS-IR) processes associated with vessels in normal adult human retina. The basement membrane of the vessels is indicated by the double headed arrows, the glia limitans by the arrowheads. (A) A large NOS-IR process (broad arrows) is in close contact with the glia limitans but was not seen to directly interact with the basement membrane of the vascular endothelium in the perivascular space. (B) Two small NOS-IR processes (broad arrows); the process on the right is located within the neuropil, in contact with the glia limitans. The process on the left is clearly located within the perivascular space.

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Figure 5.3 Substance P-immunoreactive (SP-IR) terminals associated with blood vessels in whole mounts of human retina. (A) Several terminal clusters of SP-IR varicosities (broad arrows), associated with a medium calibre vessel (bv) in the ganglion cell layer (GCL) of human retina. Individual clusters appear to be derived from different nerve fibres (thin arrows) arising from different cells. (B) A medium calibre vessel in the GCL with three side arm branches and related SP-IR cell processes (thin arrows) and a single terminal cluster of SP-IR varicosites (broad arrow). The SPimmunoreactive fibres and the SP-IR varicosity appear to be separated from the vascular endothelium by the perivascular space (double-headed arrow). (C) A single

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adjacent to a large-calibre vessel in the ganglion cell layer. The processes of the cell invest the blood vessel (bv) although no SP-IR varicosities are present. (B) A rare example of a SP-IR soma and processes which course along the length of the vessel and terminate in clusters of SP-IR varicosites (towards the left and right extremities of the micrograph). Other varicosites are also indicated (broad arrows). Note that not all of the SP-IR plexus is derived from this cell.

Figure 5.5 An optical section taken using dual-scanning confocal microscopy of a major retinal vessel in a whole mount of adult human retina, double immunolabelled with anti-glial fibrillary acidic protein (GFAP; green) and anti-substance P (SP; red). The plane of focus is the ganglion cell/nerve fibre layer interface. Astrocyte processes (AP) can be seen running approximately parallel to the nerve fibres and investing the retinal vessels, forming the perivascular glia limitans (GL). Some SP-immunoreactive somata can be seen (asterisks). In addition, SP-immunoreactive varicosities can be seen, apparently embedded in the glia limitans (broad arrows). At no time do the SP-immunoreactive varicosites appear to make contact with the vascular endothelium (VE) which is separated from the glia limitans by the perivascular space.

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Visualisation of the SP-IR varicosities, by light, confocal and electron microscopy, indicate that SP-IR terminals are separated from the vascular endothelium, and the juxtaposed pericytes and/or smooth muscle cells, by the collagen-containing perivascular space (Figures 5.3–5.6). In addition, both confocal and electron microscopy suggest that SP-IR varicosities are associated with the perivascular glia limitans of the retinal vessels (Figures 5.5 and 5.6).

While these observations provide no data concerning the function of SP-IR varicosities associated with the retinal vasculature, consideration of the known functional roles of SP in the peripheral and central nervous system suggests at least two possibilities. The first is modulation of cytokines, or other inflammatory-associated substances, in microglial cells associated with the glia limitans and/or perivascular space (Martin et al., 1993) which, in turn, may regulate barrier function. The second possibility, more germane to

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this review, is for a role in autoregulation of retinal blood flow. While activation of autonomic centres has the effect of directing major changes in blood flow, neuropeptides (along with NO) have been proposed to have a role in the rapid modification of vessel tone through autoregulation in response to local demands (Edvinsson, 1985; Said, 1987; Vincent, et al., 1992). In this context SP, particularly in association with the more potent CGRP, can act as a potent vasodilator (McCulloch, et al., 1986; Vincent, et al., 1992). The SP-IR terminals described here which associate with the glia limitans of human retina appear to be derived from intrinsic retinal cells and may represent an anatomical basis for one aspect of autoregulation of the retinal vasculature.

Finally, a recent study has also suggested a role for peptidergic neurons in control of blood flow. Within the human retina thyrotropin-like immunoreactivity has been observed in ganglion cells (Fernandez-Trujillo, Prada and Verastegui, 1996), these authors suggesting that peptides released from terminals associated with blood vessels may be involved in regulating vessel diameter and flow. The data, however, remains speculative and precise association needs to be proved before firm conclusions can be made.

The evidence, therefore, for intrinsic innervation of retinal vessels is now very compelling and provides an anatomical platform for one component of autoregulation of retinal blood flow.

CONCLUSIONS

While there is considerable evidence in support of the existence of neural mechanisms to regulate choroidal blood flow, including specialisation of these mechanisms to modulate flow to the fovea, evidence for neural substrates which mediate regulation of retinal blood flow is less readily available. There is good evidence in the literature indicating innervation of the central retinal artery, but it appears that there is no extrinsic innervation to the retinal vessels themselves. We have reviewed the limited literature describing candidate neurons intrinsic to the retina with processes which appear to make specific contact with the basement membrane of the retinal vessels and provided additional information concerning the nature of those associations.

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endothelial cell junction is indicated by the thin arrow. SPimmunoreactive terminals, labelled with electron dense, silver-enhanced immunogold particles, are indicated (broad arrows) adjacent to the glia limitans, within the retinal parenchyma. (B) SP terminals are partially separated from the perivascular space by the glia limitans. The large terminal to the right of the micrograph is partially invested by cell processes (presumably astrocytes) and in some locations directly in contact with the collagenous matrix of the perivascular space (asterisk). Scale bars=1 µm.

Whether this data is sufficient to confirm the presence of intrinsic neuronal regulation of retinal blood flow remains to be seen. Certainly, further work is required to integrate physiological and cell biological studies to demonstrate the ability of amacrine cells processes to bring about alterations in vascular tone, either through direct contraction of the endothelial cell or through perivascular-associated cells such as pericytes. The retinal vasculature is essentially devoid of an internal elastic lamina and muscularis raising the intriguing possibility that the tone of the capillary network is being controlled by pericytes. This is supported by the observation that pericytes express neurotransmitter receptors (Ferrari-Dileo, Davis and Anderson, 1991) are contractile (Kelley et al., 1987; Hirschi and D’Amore, 1996) and are present in greater numbers in the retina than in other organs, including the brain. The heterogeneity of the retinal vasculature, and indeed of the retinal endothelium (Yu et al., 1997), is another area of interest that may be of great significance in the local regulation of vascular flow and permeability.

REFERENCES

Ahmed, J., Braun, R.D., Dunn, R. and Linsenmeier, R.A. (1993). Oxygen distribution in macaque retina. Investigative Ophthalmology and Visual Science, 34, 516–521.

Bill, A. and Nilsson, S.F.E. (1985). Control of ocular blood flow. Journal of Cardiovascular Pharmacology, 7 (Suppl), S96–S102.

Bill, A. and Sperber, G.O. (1990). Control of retinal and choroidal blood flow. Eye, 4, 319–325.

Braunagel, S.C., Xiao, J.G. and Chiou, G.C.Y. (1988). The potential role of adenosine in regulating blood-flow in the eye. Journal of Ocular Pharmacology, 4, 61–73.

Brecha, N.C., Hendrickson, A., Floren, I. and Karten, H.J. (1982). Localization of substance P-like immunoreactivity within the monkey retina. Investigative Ophthalmology and Visual Science, 23, 147–153.

Brown, S.M. and Jampol, L.M. (1996). New concepts of regulation of retinal vessel tone.

Archives of Ophthalmology, 114, 199–204.

Campochiaro, P.A. and Sen, H.A. (1989). Adenosine and its agonists cause retinal vasodilation and hemorrhages. Implications for ischemic retinopathies. Archives of Ophthalmology, 107, 412–416.

Nervous control of the eye 200

Crosson, C.E., DeBenedetto, R. and Gidday, J.M. (1994). Functional evidence for retinal adenosine receptors. Journal of Ocular Pharmacology, 10, 499–507.

Cuenca, C. and Kolb, H. (1998). Circuitry and role of substance P-immunoreactive neurons in the primate retina. Journal of Comparative Neurology, 393, 439–456.

Cuenca, C., De Juan, J. and Kolb, H. (1995). Substance P-immunoreactive neurons in the human retina. Journal of Comparative Neurology, 356, 491–504.

Diaz, C.M., Penfold., P.L. and Provis, J.M. (1998). Modulation of the resistance of a human endothelial cell line by human retinal glia. Australian and New Zealand Journal of Ophthalmology, 26 (Suppl.), S62.

Donati, G., Pournaras, C.J., Munoz, J.-L., Poitry, S., Poitry-Yamate, C.L. and Tsacopoulos, M. (1995). Nitric oxide controls arteriolar tone in the retina of the miniature pig. Investigative Ophthalmology and Visual Science, 36, 2228–2237.

Edvinsson, L. (1985). Functional role of perivascular peptides in the control of cerebral circulation. Trends in the Neurosciences, 8, 126–131.

Fernandez-Trujillo, F.J., Prada, A. and Verastegui, C. (1996). Thyrotropin-like immunoreactivity in human retina: immunoreactive co-localization in ganglion cells and perivascular fibres. Neurochemistry International, 28, 381–384.

Ferrari-Dileo, G., Davis, E.B. and Anderson, D.R. (1991). Cholinergic binding sites in pericytes isolated from retinal capillaries. Blood Vessels, 28, 542–546.

Flugel, C., Tamm, E.R., Mayer, B. and Lutjen-Drecoll, E. (1994). Species differences in choroidal vasodilative innervation: evidence for specific intrinsic nitridergic and VIPpositive neruons in the human eye. Investigative Ophthalmology and Visual Science,

35, 592–599.

Flugel-Koch, C., Kaufman, P. and Lutjen-Drecoll, E. (1994). Association of a choroidal ganglion cell plexus with the fovea centralis. Investigative Ophthalmology and Visual Science, 35, 4268–4272.

Funk, R.H.W. (1997). Blood supply of the retina. Ophthalmic Research, 29, 320–325. Gidday, J.M. and Park, T.S. (1993). Adenosine-mediated autoregulation of retinal

arteriolar tone in the piglet. Investigative Ophthalmology and Visual Science, 34,

2713–2719.

Greenwood, J. (1992). Experimental manipulation of the blood-brain and blood-retinal barriers. In: Physiology and pharmacology of the blood-brain barrier, edited by M.W.B.Bradbury. Handbook of Experimental Pharmacology, 103, 459–486.

Greenwood, J., Bamforth, S., Wang, Y. and Devine, L. (1995). The blood-retinal barrier in immune-mediated diseases of the retina. In: New concepts of a blood-brain barrier, edited by J.Greenwood, D.Begley and M.Segal, pp 315–326. London: Plenum Press.

Haefliger, I.O., Meyer, P., Flammer, J. and Luscher, T.F. (1994). The vascular endothelium as a regulator of the ocular circulation: a new concept in ophthalmology?

Survey of Ophthalmology, 39, 123–132.

Hirschi, K.K. and D’Amore, P. (1996). Pericytes in the microvasculature. Cardiovascular Research, 32, 687–698.

Janzer, R.C. and Raff, M.C. (1987). Astrocytes induce blood-brain barrier properties in endothelial cells. Nature, 352, 253–257.

Kelley, C., D’Amore, P., Hechtmann, H.B. and Shepro, D. (1987). Microvascular pericyte contractility in vitro: Comparison with other cells of the vascular wall. Journal

Evidence for the intrinsic innervation of retinal vessels 201

of Cell Biology, 104, 483–490.

Kitamura, Y., Okamura, T., Kani, K and Toda, N. (1993). Nitric oxide-mediated retinal arteriolar and arterial dilatation induced by substance P. Investigative Ophthalmology and Visual Science, 34, 2859–2865.

Klooster, J., Beckers, H.J.M, ten Tusscher, M.P.M., Vrensen, G.F.J.M., van der Want, J.J.L. and Lamers, W.P.M.A. (1996). Sympathetic innervation of the rat choroid: an autoradiographic tracing and immunohistochemical study. Ophthalmic Research, 28, 36–43.

Kolb, H., Fernandez, E., Ammermuller, J. and Cuenca, N. (1995). Substance P: a neurotransmitter of amacrine and ganglion cells in the vertebrate retina. Histology and Histopathology, 10, 947–68.

Komai, K., Miyazaki, S., Onoe, S., Shimo-Oku, M. and Hishida, S. (1995). Vasomotor nerves of vessels in the human optic nerve. Acta Ophthalmologica Scandinavica, 73, 512–516.

Kumagai, N., Yuda, K., Kadota, T., Goris, R.C. and Kishida, R. (1988). Substance P-like immunoreactivity in the central retinal artery of the rabbit. Experimental Eye Research, 46, 591–596.

Laties, A.M. (1967). Central retinal artery innervation. Archives of Ophthalmology, 77, 405–409.

Li, H.-B. and Lam, D.M.-K. (1990). Localization of neuropeptide-immunoreactive neurons in the human retina. Brain Research, 522, 30–36.

Marshak, D.W. (1989). Peptidergic neurons of the macaque monkey retina. Neuroscience Research, 10, S117– S130.

Martin, F.C., Anton., P.A., Gornbein, J.A. and Merrill, J.E. (1993). Production of interleukin-1 by microglia in response to substance P: Role for non-classical NK-1 receptor. Journal of Neuroimmunology, 42, 53–60.

McCulloch, J., Uddman, R., Kingman, T.A. and Edvinsson, L. (1986). Calcitonin generelated peptide: functional role in cerebrovascular regulation. Proceeding of the National Academy of Sciences USA., 83, 5731–5735.

Nilsson, S.F.E. (1996). Nitric oxide as a mediator of parasympathetic vasodilation in ocular and extraocular tissues in the rabbit. Investigative Ophthalmology and Visual Science, 37, 2110–2119.

Nuzzi, R., Gugleilmone, R. and Grignolo, F.M. (1995). Fluorescence histochemical demonstration of adrenergic terminations in the human choroid. European Journal of Ophthalmology, 5, 251–258.

Owman, C., Edvinsson, L. and Nielsen, K.C. (1974). Autonomic neuroreceptor mechanisms in brain vessels. Blood Vessels, 11, 2–31.

Penfold, P.L. and Provis, J.M. (1998). The neurovascular interface in human retina.

Experimental Eye Research., 67 (Suppl), 94.

Provis, J.M. and Mitrofanis, J. (1990). NADPH-diaphorase neurones of human retinae have a uniform topographical distribution. Visual Neuroscience, 4, 619–623.

Provis, J.M., Yip, J. and Penfold, P.L. (1992). Substance P in the human retina.

Proceedings of the Australian Neuroscience Society, 3, 62.

Provis, J.M., Penfold, P.L., Edwards, A.J. and van Driel, D. (1995). Human retinal microglia: Expression of immune markers and relationship to the glia limitans. Glia,

Nervous control of the eye 202

14, 243–256.

Raub, T.J., Luentzel, S.L. and Sawada, G.A. (1992). Permeability of bovine brain microvessel endothelial cells in vitro; barrier tightening by a factor released from astroglioma cells. Experimental Cell Research, 199, 330–340.

Roufail, E., Stringer, M. and Rees, S. (1995). Nitric oxide synthase immunoreactivity and NADPH diaphorase staining are co-localised in neurons closely associated with the vasculature in rat and human retina. Brain Research, 684, 36–46.

Ruskell, G.L. (1971). Facial parasympatheitc innervation of the choroid blood vessels in monkeys. Experimental Eye Research, 12, 166–172.

Said, S.I. (1987) Neuropeptides in cardiovascular regulation. In: Brain Peptides and Catcholamines in Cardiovascular Regulation, edited by J.P.Buckley and C.M.Ferrario, pp. 93–107. New York: Raven Press.

Sen, H.A. and Campochiaro, P.A. (1989). Intravitreal injection of adenosine or its agonists causes breakdown of the blood-retinal barrier. Archives of Ophthalmology, 107, 1364–1367.

Stjernschantz, J. and Bill, A. (1980). Vasomotor effects of facial nerve stimulation: noncholinergic vasodilation in the eye. Acta Physiologica Scandinavica, 109, 45–50.

Stone, J. and Dreher, Z. (1987). Relationship between astrocytes, ganglion cells and vasculature of the retina. Journal of Comparative Neurology, 255, 35–49.

Toda, N., Kitamura, Y. and Okamura, T. (1994). Role of nitroxidergic nerve in dog retinal arterioles in vivo and arteries in vitro. American Journal of Physiology, 266,

H1985–H1992.

Verstappen, A., Toussaint, D., Lotstra, F. and Van der Haeghen, J.J. (1986). Club endings containing substance P-like immunoreactivity in human retinal vessels: whole mount preparation. Neurochemistry International, 8, 377–380.

Vincent, M.B., White, L.R., Elsas, T., Qvigstad, G. and Sjaastad, O. (1992). Substance P augments the rate of vasodilation induced by calcitonin gene-related peptide in porcine ophthalmic artery in vitro. Neuropeptides, 22, 137–141.

Ye, X., Laties, A. and Stone, R. (1990). Peptidergic innervation of the retinal vasculature and optic nerve head. Investigative Ophthalmology and Visual Science, 31, 1731–1737.

Yu, P.K., Yu, D-Y., Alder, V.A., Seydel, U., Su, E-N. and Cringle, S.J. (1997). Heterogeneous endothelial cell structure along the porcine retinal microvasculature.

Experimental Eye Research, 65, 379–389.